C-C chemokine receptor 3 proteins

ABSTRACT

The present invention relates to isolated and/or recombinant C—C Chemokine Receptor 3 (CKR-3, CCR3) proteins or polypeptides.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/963,656, filed Nov. 3, 1997, which is a division of U.S. applicationSer. No. 08/720,565, filed Sep. 30, 1996 (U.S. Pat. No. 6,537,764 B1)which is a continuation-in-part of International ApplicationPCT/US96/00608 (designating the United States), with an Internationalfiling date of Jan. 19, 1996, which is a continuation-in-part of U.S.application Ser. No. 08/375,199, filed Jan. 19, 1995 (U.S. Pat. No.6,806,061 B1), the teachings of which are each incorporated herein byreference in their entirety.

GOVERNMENT SUPPORT

Work described herein was supported in whole or in part by a U.S.government grant. The U.S. government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Chemokines, also referred to as intecrines, are soluble, low molecularweight members of the cytokine family which have chemoattractantfunction. Chemokines are capable of selectively inducing chemotaxis ofthe formed elements of the blood (other than red blood cells), includingleukocytes such as monocytes, macrophages, eosinophils, basophils, mastcells, and lymphocytes, such as T cells, B cells, and polymorphonuclearleukocytes (neutrophils)). In addition to stimulating chemotaxis, otherchanges can be selectively induced by chemokines in responsive cells,including changes in cell shape, transient rises in the concentration ofintracellular free calcium ([Ca²⁺]_(i)), granule exocytosis, integrinupregulation, formation of bioactive lipids (e.g., leukotrienes) andrespiratory burst, associated with leukocyte activation. Thus, thechemokines are early triggers of the inflammatory response, causinginflammatory mediator release, chemotaxis and extravasation to sites ofinfection or inflammation.

The chemokines characterized to date are related in primary structure.They share four conserved cysteines, which form disulphide bonds. cDNAcloning and biochemical characterization of several chemokines hasrevealed that the proteins have a leader sequence of 20-25 amino acids,which is cleaved upon secretion to yield a mature protein ofapproximately 92-99 amino acids. Based on the conserved cysteine motif,the family is divided into two branches, designated as the C—Cchemokines (β chemokines) and the C—X—C chemokines (α chemokines), inwhich the first two conserved cysteines are adjacent or are separated byan intervening residue, respectively. Baggiolini, M. and C. A. Dahinden,Immunology Today, 15: 127-133 (1994)).

The C—X—C chemokines include a number of chemoattractants which arepotent chemoattractants and activators of neutrophils, such asinterleukin 8 (IL-8), PF4 and neutrophil-activating peptide 2 (NAP-2).The C—C chemokines include molecules such as human monocyte chemotacticproteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation,Normal T Expressed and Secreted), and the macrophage inflammatoryproteins 1α and 1β (MIP-1α and MIP-1β), which have been characterized aschemoattractants and activators of monocytes or lymphocytes, but do notappear to be chemoattractants for neutrophils. For example, recombinantRANTES is a chemoattractant for monocytes, as well as for memory T cellsin vitro (Schall, T. J. et al., Nature, 347: 669-671 (1990)). Morerecently a chemokine called lymphotactin with a single cysteine pair inthe molecule has been identified which attracts lymphocytes (Kelner, G.S., et al., Science, 266: 1395-1359 (1994)).

The C—C chemokines are of great interest because of their potential rolein allergic inflammation. For example, MCP-1 induces exocytosis of humanbasophils, resulting in release of high levels of inflammatorymediators, such as histamine and leukotriene C₄. Similarly, there isgreat interest in the receptors for the C—C chemokines, which triggerthese cellular events in response to chemokine binding. A receptor forC—C chemokines has recently been cloned and is reported to bind MIP-1αand RANTES. Accordingly, this MIP-1α/RANTES receptor was designated C—Cchemokine receptor 1 (CKR-1; Neote, K. et al., Cell, 72: 415-425 (1993);Horuk, R. et al., WO 94/11504, published May 26, 1994; Gao, J. -I. etal., J Exp. Med., 177: 1421-1427 (1993)). An MCP-1 receptor has alsobeen cloned (Charo, I. F. et al., Proc. Natl. Acad. Sci. USA, 91: 2752(1994)). This receptor, designated CKR-2, is reported to bind MCP-1 withhigh affinity and MCP-3 with lower affinity (Charo, I. F., et al., Proc.Natl. Acad. Sci. USA, 91: 2752-2756 (1994)). CKR-2 has been shown toexist in two isoforms resulting from the use of an alternative splicesite in isoform A producing a distinct cytoplasmic tail. Isoform B,which is not spliced in this region, has been shown to be a functionalreceptor for MCP-1 and MCP-3 in binding and signal transduction assays(Charo, I. F., et al., Proc. Natl. Acad. Sci. USA, 91: 2752-2756 (1994);Myers, S. J., et al., J. Biol. Chem., 270: 5786-5792 (1995)). Morerecently, a new receptor called CKR-4 has been described; cRNA from thisreceptor was reported to produce a Ca²⁺ activated chloride current inresponse to MCP-1, MIP-1α, and RANTES when injected in to X. laevisoocytes (Power, C. A., et al., J. Biol. Chem., 270: 19495-19500 (1995)).

The MCP-1 receptor (CKR-2) and C—C chemokine receptor 1 are predicted tobelong to a superfamily of seven transmembrane spanning G-proteincoupled receptors (Gerard C., and Gerard, N. P., Annu. Rev. Immunol.,12: 775-808 (1994); Gerard C., and Gerard N. P., Curr. Opin. Immunol.,6: 140-145 (1994)). This family of G-protein coupled (serpentine)receptors comprises a large group of integral membrane proteins,containing seven transmembrane-spanning regions. The ligands of thesereceptors include a diverse group of molecules, including small biogenicamine molecules, such as epinephrine and norepinephrine, peptides, suchas substance P and neurokinins, and larger proteins, such as chemokines.The receptors are coupled to G proteins, which are heterotrimericregulatory proteins capable of binding GTP and mediating signaltransduction from coupled receptors, for example, by the production ofintracellular mediators.

The cloning and sequencing of two IL-8 receptor cDNAs reveals that theseC—X—C receptor proteins also share sequence similarity with seventransmembrane-spanning G protein-coupled receptor proteins (Murphy P. M.and H. L. Tiffany, Science, 253: 1280-1283 (1991); Murphy et al., WO93/06299; Holmes, W. E. et al., Science, 253: 1278-1280 (1991)).Additional receptors for chemotactic proteins such as anaphylatoxin C5aand bacterial formylated tripeptide fMLP have been characterized bycloning and been found to encode receptor proteins which also sharesequence similarity to these seven transmembrane-spanning proteins(Gerard, N. P. and C. Gerard, Nature, 349: 614-617 (1991); Boulay, F. etal., Biochemistry, 29: 11123-11133 (1990)). Although a number of otherproteins with significant sequence similarity and similar tissue andleukocyte subpopulation distribution to known chemokine receptors havebeen identified and cloned, the ligands for these receptors remainundefined. Thus, these proteins are referred to as orphan receptors.

The isolation and characterization of additional genes and the encodedreceptors, and the characterization of the corresponding ligands, isessential to an understanding of the interaction of chemokines withtheir target cells and the events stimulated by this interaction,including chemotaxis and cellular activation of leukocytes.

SUMMARY OF THE INVENTION

The present invention relates to isolated and/or recombinant nucleicacids which encode a mammalian (e.g., human) receptor protein designatedC—C Chemokine Receptor 3 (CKR-3 or CCR3). The invention further relatesto recombinant nucleic acid constructs, such as plasmids or retroviralvectors, which contain a nucleic acid which encodes a receptor proteinof the present invention, or portions of said receptor. The nucleicacids and constructs can be used to produce recombinant receptorproteins. In another embodiment, the nucleic acid encodes an antisensenucleic acid which can hybridize with a second nucleic acid encoding areceptor of the present invention, and which, when introduced intocells, can inhibit the expression of receptor.

Another aspect of the present invention relates to proteins orpolypeptides, referred to herein as isolated, recombinant mammalianCKR-3 receptors. The recombinant CKR-3 receptors or polypeptides can beproduced in host cells as described herein. In one embodiment, areceptor protein is characterized by high affinity binding of one ormore chemokines, such as eotaxin, RANTES and/or MCP-3, and/or theability to stimulate a (one or more) cellular response(s) (e.g.,chemotaxis, exocytosis, release of one or more inflammatory mediators).

Antibodies reactive with the receptors can be produced using thereceptors or portions thereof as immunogen or cells expressing receptorprotein or polypeptide, for example. Such antibodies or fragmentsthereof are useful in therapeutic, diagnostic and research applications,including the purification and study of the receptor proteins,identification of cells expressing surface receptor, and sorting orcounting of cells.

Also encompassed by the present invention are methods of identifyingligands of the receptor, as well as inhibitors (e.g., antagonists) orpromoters (agonists) of receptor function. In one embodiment, suitablehost cells which have been engineered to express a receptor protein orpolypeptide encoded by a nucleic acid introduced into said cells areused in an assay to identify and assess the efficacy of ligands,inhibitors or promoters of receptor function. Such cells are also usefulin assessing the function of the expressed receptor protein orpolypeptide.

According to the present invention, ligands, inhibitors and promoters ofreceptor function can be identified and further assessed for therapeuticeffect. Ligands and promoters can be used to stimulate normal receptorfunction where needed, while inhibitors of receptor function can be usedto reduce or prevent receptor activity. Thus, the present inventionprovides a new strategy of anti-inflammatory therapy, useful in avariety of inflammatory and autoimmune diseases, comprisingadministering an inhibitor of receptor function to an individual (e.g.,a mammal). In contrast, stimulation of receptor function byadministration of a ligand or promoter to an individual provides a newapproach to selective stimulation of leukocyte function, which isuseful, for example, in the treatment of parasitic infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrates the nucleotide sequence determined from agenomic clone encoding a human CKR-3 protein also referred to as Eos L2receptor (SEQ ID NO:1), and the predicted amino acid sequence of theprotein encoded by the open-reading frame (SEQ ID NO:2).

FIGS. 2A-2C illustrates the nucleotide sequence determined from thecDNAs encoding a human CKR-3 receptor (SEQ ID NO:3), and the predictedamino acid sequence of the protein encoded by the open-reading frame(SEQ ID NO:4).

FIG. 3 is an illustration of one type of transendothelial chemotaxisassay. A culture insert is placed into a container, such as a well in a24-well plate, creating a first and second chamber within the well.ECV304 endothelial cells are grown in a monolayer on the polycarbonatemembrane on the inner side of the insert. Cells to be assessed for aresponse to a substance (e.g., a chemokine) are introduced into the topchamber and the substance is introduced into the bottom chamber.Chemotaxis can be assessed by detecting cells which migrate through theendothelial layer into the bottom chamber, by removing the insert anddetecting or counting cells by a suitable method. For example, cells inthe bottom chamber can be collected and assessed by flow cytometry(e.g., FACS analysis, light scattering).

FIG. 4 is a histogram illustrating the chemotaxis of human eosinophilsin response to various chemokines. Human eosinophils were purified usinga standard protocol, and assessed by microscopy for their response tovarious chemokines in a 24 well transendothelial chemotaxis assay (cellsper high power field (HPF).

FIGS. 5A-5I are an illustration of a FACS analysis of various clones ofL1-2 pre-B cells transfected with Eos L2. Cells from over 200 cloneswere stained with M2 anti-FLAG Mab followed by anti-mouse Ig-FITC.(Y-axis, number of cells; X-axis, fluorescence). In the negative control(PAUL 001), transfected cells were stained with an irrelevant antibody.

FIG. 6 is a histogram illustrating the binding of RANTES and MIP-1α tohuman eosinophils. Purified normal human eosinophils were incubated with0.1 nM ¹²⁵I-labeled MIP-1α or RANTES (“Hot”) in the presence or absenceof various cold chemokines (MIP-1α, RANTES, IL-8, MCP-1, MCP-3) at 250nM.

FIG. 7 is a graph illustrating inhibition of the binding of ¹²⁵I labeledRANTES to human eosinophils by various cold chemokines (RANTES, MIP-1α,MCP-1 and MCP-3). Human eosinophils were incubated with 0.1 nMradiolabeled RANTES and the indicated concentrations of cold chemokines.The data plotted are the means and standard deviations of duplicates foreach sample.

FIG. 8 is a histogram illustrating the binding of 0.1 nM ¹²⁵I-labeled(“Hot”) RANTES or 0.1 nM ¹²⁵1I-labeled (“Hot”) MCP-3 to Eos L2 infectedSF9 cells (cpm, counts per minute). (From left to right: Hot Rantesonly; Hot Rantes+Cold Rantes; Hot MCP-3 only; Hot MCP-3+cold MCP-3).

FIGS. 9A-9D are graphs illustrating CKR-3 expression on leukocytes asdetermined using MAb LS26-5H12 and flow cytometry. Leukocyte subsetswere stained with anti-CKR-3 MAb LS26-5H12 (solid lines) or an IgG₁isotype-matched control antibody (MOPC-21) (dotted lines). FIG. 9A,eosinophils; FIG. 9B, T Cells; FIG. 9C, monocytes; FIG. 9D, neutrophils.Dead cells were excluded based on propidium iodide staining.

FIGS. 10A-10C are graphs illustrating cell surface staining of L1.2cells transiently transfected with a CKR-3 receptor (FIG. 10A),mock-transfected L1.2 control cells (FIG. 10B), or cell line E5 (astable L1.2 CKR-3 transfectant) (FIG. 10C) with an anti-CKR-3 monoclonalantibody (LS26-5H12, solid line). Background staining with controlmonoclonal antibody MOPC-21 is also shown (dotted lines).

FIGS. 11A-11D are graphs illustrating the results of competitive ligandbinding of radiolabeled human eotaxin to the E5 cell line (a stable L1-2cell line transfected with a CKR-3 receptor; FIG. 11A) or to humaneosinophils (FIG. 11B). Cells were incubated with 0.6 nM ¹²⁵I-labeledeotaxin and various concentrations of unlabeled eotaxin (◯), RANTES (▴),or MCP-3 (□). After 60 minutes at room temperature, cell pellets werewashed and counted. Scatchard plots of unlabeled eotaxin competitionwere calculated from the data (FIG. 11C, E5 cell line; FIG. 11D,eosinophils).

FIG. 12 is a histogram illustrating the inhibition by various chemokinesof human eotaxin binding to the E5 cell line. E5 cells (stableL1-2/CKR-3 transfectants) were incubated with 0.6 nM radiolabeledeotaxin and 250 nM unlabeled chemokines or no competitor as indicated.

FIGS. 13A-13C are histograms illustrating chemotaxis of L1.2 cells andL1.2 receptor transfectants. 1×10⁶ cells of the E5 cell line (stableL1-2/CKR-3 transfectants) (FIG. 13A), the parental L1.2 cell line (FIG.13B), or an IL-8 RB L1.2 receptor transfectant line LSLW-2 (FIG. 13C)were placed in the top chamber and chemokines placed in the bottomchamber at the concentrations specified. Migration was allowed for 4hours and cells migrating to the bottom chamber were counted. All assayswere performed in duplicate and the results representative of at leastthree separate experiments. Chemokines are listed along the x-axis,number of cells migrated along the y-axis, and concentration ofchemokine along the z-axis.

FIGS. 14A-14B are graphs illustrating the chemotactic response ofeosinophils from two different individuals. The response resembles thatof CKR-3 L1.2 transfectants. Donor to donor variation of chemotacticresponses of eosinophils to eodaxin, RANTES, MCP-3, and MIP-1α wasobserved. Eosinophils were purified from blood, and assessed for theirchemotactic response to various concentrations of chemokines. Values arefrom a representative experiment of at least 4 performed, using the sametwo blood donors.

FIG. 15 is a graph illustrating the binding of ¹²⁵I-labeled RANTES tomembranes from a stable cell line (A31-293-20) obtained by transfecting293 cells with the A31 cDNA clone (square with central dot) as comparedwith binding to membranes from untransfected 293 cells (filled circles).

FIG. 16 is a histogram illustrating the binding of ¹²⁵I-labeled MCP-3 toa membranes from a stable cell line (A31-293-20) obtained bytransfecting 293 cells with the A31 cDNA clone as compared with bindingto membranes from untransfected 293 cells. Binding of labeled MCP-3 tomembranes from transfected (A31-20) or untransfected (UT293) cells wasdetermined in the absence of cold MCP-3 (0 nM) or in the presence ofcold MCP-3 (100 nM).

FIG. 17 is a histogram illustrating the specificity of binding, whichwas assessed by determining the amount of bound ¹²⁵I-labeled MCP-3 whichcould be displaced by cold MCP-3 from membranes of transfected (A31-20)or untransfected (UT293) cells.

FIG. 18A is a FACs profile of the fluorescence intensity of stable L1.2transfectants expressing either CCR1, CCR2, CCR3, CCR4, CCR5, CXCR1(IL-8 RA), or CXCR2 (IL-8 RB) which were stained with anti CCR3 mAb7B11. Negative control staining for all the L1.2 transfectants (notshown) resembled the staining shown for 7B11 on CCR1 transfectants.

FIG. 18B is a FACs profile of human eosinophils, lymphocytes, T cellblasts, monocytes, and granulocytes stained with mAb 7B11. Stainingprofiles were representative of at least 4 experiments.

FIG. 18C is a histogram illustrating binding of radiolabeled humaneotaxin, RANTES, MCP-2, or MCP-3 to L1.2 CCR3 or CCR1 transfectants, andinhibition by mAb 7B11 or cold chemokines. Cells were incubated with 0.1nM ¹²⁵I-labeled eotaxin, RANTES, or MCP-3, and either 50 μl of 100 μg/mlof irrelevant mAb (MOPC 21), mAb 7B11, or 250 nM cold chemokine. After60 minutes at room temperature, cell pellets were washed and counted.

FIG. 19 is a graph illustrating inhibition of binding of radiolabeledeotaxin, RANTES, and MCP-3 to human eosinophils by mAb 7B11. Humaneosinophils were incubated with 0.1 nM ¹²⁵I-labeled-eotaxin, -RANTES, or-MCP-3, and various concentrations of mAb 7B11. After 60 minutes at roomtemperature, cell pellets were washed and counted. Data was analyzed byKaleidaGraph, which calculated an IC50 of eotaxin of 25.7 ng/ml, forRANTES of 13.7 ng/ml, and for MCP-3 of 18.8 ng/ml. The level ofinhibition using 250 nM cold chemokine is shown at the bottom left ofthe plot: ◯ eotaxin, □ RANTES, and Δ MCP-3.

FIG. 20A is a graph illustrating the dose response of mAb 7B11inhibition of eosinophil chemotaxis to eotaxin. The level of backgroundmigration of cells (no chemokine) is shown by the □ symbol (bottom leftof the plot).

FIG. 20B is a histogram illustrating inhibition of eosinophil chemotaxisto various chemoattractants by 5 μg or 20 μg/ml of 7B11 mAb. For theexperiments shown in both 20A and 20B, 1×10⁶ human eosinophils wereplaced in the top chamber of the transwell and 10 nM of chemokine wasplaced in the bottom chamber. Various concentrations of 7B11 mAb wereplaced in the top well. After 1.5 hours the cells migrating to thebottom chamber were counted using flow cytometry. The results arerepresentative of at least four separate experiments.

FIGS. 21A-21J are a series of tracings illustrating that mAb 7B11inhibits [Ca²⁺]i by human eosinophils in response to eotaxin, RANTES,MCP-2, MCP-3 and MCP-4. Human eosinophils were labeled with Fura-2, andstimulated sequentially with mAb (A), followed 40 sec later with theindicated chemokine (B), and 100 sec following that with C5a (C).[Ca²⁺]i fluorescence changes were recorded using a spectrofluorimeter.The tracings are representative of five separate experiments, performedwith eosinophils from different donors. In the top panels, an irrelevantcontrol mAb (MOPC-21) was used, and in the bottom panels, mAb 7B11.Antibodies were used at a final concentration of 6.4 μg/ml Chemokineswere used at: eotaxin, 10 nM, RANTES, 20 nM, MCP-2, 200 nM, MCP-3, 200nM, MCP-4, 10 nM. C5a was used at 400 pM.

FIG. 22A is a FACs profile illustrating IL-8 receptor expression onfreshly isolated eosinophils from a healthy individual. Eosinophils werestained with mAbs to CXCR1 (solid line), CXCR2 (dotted line) or acontrol mAb (shaded), and were analyzed by flow cytometry.

FIG. 22B is a FACs profile illustrating IL-8 receptor expression on IL-5treated eosinophils. Eosinophils cultured with IL-5 for 5 days werestained with mAbs, as in FIG. 22A.

FIG. 22C is a FACs profile illustrating IL-8 receptor expression oneosinophils isolated from an eosinophilic individual, and stained withmAbs, as in FIGS. 22A and 22B.

FIG. 22D are tracings illustrating inhibition of [Ca²⁺]i of day 5 IL-5primed eosinophils to various chemokines by mAb 7B11. Methods were thesame as those described in the legend of FIG. 21. The mAbs andchemokines used were: 1. control mAb, eotaxin, C5a; 2. 7B11, eotaxin,C5a; 3. control mAb, RANTES, C5a; 4. 7B11, RANTES, C5a; 5. control mAb,IL-8, C5a; 6. 7B11, IL-8, C5a. The results are representative of atleast three separate experiments.

FIG. 23A is a histogram illustrating blockade of eotaxin-, RANTES- andMCP-3-induced eosinophil peroxidase (EPO) release by monoclonal antibody7B11. Cross hatched bars indicate the amount of EPO released by either10 nM eotaxin, 100 nM eotaxin, 100 nM RANTES or 100 nM MCP-3. Black barsindicate the amount of EPO released when 10 μg/ml of 7B11 was present inthe eosinophil degranulation assay. The bar marked “blank” correspondsto a no chemokine, no antibody (buffer) control.

FIG. 23B is a histogram illustrating the effect of mAb 7B11 onC5a-induced eosinophil peroxidase release. The cross hatched barindicates the amount of EPO released by 1 nM C5a. The black barindicates the amount of EPO released when 10 μg/ml of 7B11 was presentin the eosinophil degranulation assay.

FIG. 24A is a graph illustrating eosinophil degranulation induced byeotaxin measured by release of eosinophil peroxidase (EPO) andeosinophilic cationic protein (ECP).

FIG. 24B is a graph illustrating eosinophil degranulation induced by C5ameasured by release of eosinophil peroxidase (EPO) and eosinophiliccationic protein (ECP).

FIG. 25 is a graph illustrating stimulation of peroxidase release fromeosinophils by eotaxin.

FIG. 26 is a graph illustrating stimulation of glucuronidase releasefrom eosinophils by eotaxin.

FIG. 27 is a graph illustrating stimulation of arylsulfatase B releasefrom human eosinophils by eotaxin.

FIG. 28 illustrates expression of CCR3 on eosinophil and basophils inwhole blood. Whole blood was stained with 7B11-FITC and anti-human IgEbiotin followed by Streptavidin quantum Red as described in Example 12and analyzed by flow cytometry.

FIG. 29 is a histogram illustrating histamine release by human basophilsin response to chemokines.

FIG. 30A is a graph illustrating that basophils chemotax in response toeotaxin and MCP-4.

FIG. 30B is a histogram illustrating blockade of basophil chemotaxis isin response to eotaxin and MCP-4 using anti-CCR3 mAb 7B11.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, nucleic acids encoding a novel human receptor,designated Eos L2 or C—C chemokine receptor 3 (CKR-3), also referred toherein as “CCR3”, have been isolated. Both human genomic and cDNA cloneshave been characterized. The cDNA clone was isolated from an eosinophilcDNA library constructed from eosinophils obtained from a patient withhypereosinophilic syndrome. Sequence analysis of the clones revealed agene containing an open reading frame of 1065 nucleotides encoding apredicted protein of 355 amino acids (FIGS. 1A-1D and 2A-2C; SEQ ID NOS:2 and 4), which shares amino acid sequence similarity with other C—Cchemokine receptors, which are believed to be G protein-coupledreceptors and to have a similar structure of seven transmembranespanning regions.

The predicted proteins encoded by CKR-3 genomic and cDNA clones containfour cysteine residues, one in each of the extracellular domains atpositions 24, 106, 183 and 273 (SEQ ID NOS:2 and 4). Cysteines at thesepositions are conserved in all chemokine receptors, including CKR-1, CKR2, CKR-4, IL8-RA and IL8-RB. In addition, this receptor contains anamino acid motif, DRYLAIVHA (residues 130-138) (SEQ ID NOS: 2 and 4),which is also highly conserved among C—X—C and C—C chemokine receptorsand is predicted to be intracellular. There are two consensus sites forprotein kinase C phosphorylation (Kishimoto, A., et al., J. Biol. Chem.,260: 12492-12499 (1985); Woodgett, J. R., Eur. J Biochem., 161: 177-184(1986)), one in the third intracellular loop at AA position 231, and onein the cytoplasmic tail at AA position 333. In addition, there are eightserine/threonine residues in the cytoplasmic tail, which may serve asphosphorylation sites for G-protein coupled receptor kinases such asthose isolated from neutrophils (Haribabu, B. and R. Snyderman, Proc.Natl. Acad. Sci. USA, 90: 9398 (1993)) or other related family members(Benovic, J. L., and Gomez, J., J. Biol. Chem., 268: 19521-19527 (1993);Kunapuli, P., and Benovic, J. L., Proc. Natl. Acad. Sci. USA, 90:5588-5594 (1993)). Serine/threonine rich cytoplasmic tails are also acommon feature of chemokine receptors. Unlike CKR-1, CKR-2, CKR-4,IL-8RA and IL-8RB receptors, CKR-3 does not contain sites for N-linkedglycosylation in any extracellular domain. The CKR-3 receptor protein isdistinct from C—C chemokine receptor 1, also referred to as theMIP-1α/RANTES receptor.

The nucleic acid sequences obtained from genomic and cDNA libraries wereco-linear, with the following exceptions. Upstream of the initiationcodon the two sequences diverge (at position 78 of FIG. 2A). The genomicclone appears to have an intron which separates the promoter and most ofthe 5′ untranslated region from the coding region. This genomicarrangement is similar to that found in other seventransmembrane-spanning chemoattractant receptors (Gerard, N. P., et al.,Biochemistry, 32: 1243-1250 (1993); Murphy, P. M., et al., Gene, 133:285-290 (1993)) including IL-8 RA and RB (Ahuja, S. K., et al., J. Biol.Chem., 269: 26381-89 (1994); Sprenger, H., et al., J. Biol. Chem., 269:11065-11072 (1994); Sprenger, H., et al., J. Immunol., 153: 2524-2532(1994)) and CKR-1 (Gao, J. L., et al., J. Exp. Med., 177:1421-1427(1993)). Furthermore, examination of the genomic sequence around thepoint of divergence reveals a canonical splice acceptor sequence.

Initial sequence information revealed two regions in which the cDNAsequence appeared to be shifted in frame, resulting from an insertion ofa base followed by the deletion of a base, or the deletion of a basefollowed by the insertion of a base. These alterations resulted in fourcontiguous amino acid differences in the predicted proteins at positions263-266 and 276-279, respectively. Other differences led to amino aciddifferences at positions 182, 196, 197, and 315 of the predictedproteins. The nucleotide sequence presented in SEQ ID NO:5 is aconsensus sequence, which includes regions which were sequenced in bothclones, and was constructed by simple alignment (base for base) of theinitial nucleic acid sequences. SEQ ID NO:6, in which the inital aminoacid differences between the cDNA and genomic clones are indicated byXaa, represents the predicted protein of SEQ ID NO:5. However, furthersequence analysis revealed that nucleotide sequences of the open readingframes appear to differ only at a position corresponding to nucleotides918-919 of FIG. 2B. The genomic clone has a CG at this position, whilethe cDNA clone has a GC at this position. Thus, the genomic clone codesfor threonine (ACG) at position 276 and the cDNA clone codes for serine(AGC) at position 276. The difference may be due to a sequencingambiguity, or an error introduced into the cDNA during reversetranscription. Alternatively, the conservative subsitution(serine/threonine) could be due to polymorphism between individuals.Another alternative is that the differences are due to mutation of thereceptor gene in the eosinophils of the patient from which RNA for cDNAlibrary construction was obtained.

Monoclonal and polyclonal antibodies specific for a C—C chemokinereceptor 3 of human origin were produced using an N-terminal syntheticpeptide of the receptor. FACS (fluorescence activated cell sorting)analysis using one of the monoclonal antibodies (LS26-5H12) revealedsignificant expression of this receptor on human eosinophils, but not onleukocytes including monocytes, neutrophils, lymphocytes, T cells, Tcell blasts (produced by activation with CD3 MAb) (FIGS. 9A-9D). Thispattern of expression was confirmed by Northern analysis with RNA fromhighly purified leukocyte subsets. However, in some experiments, CKR-3mRNA or receptor was detected in T lymphocytes; accordingly, it ispossible that CKR-3 is expressed on a subset of T lymphocytes (Example5). In addition, as described herein, a monoclonal antibody specific forthe C—C chemokine receptor 3 of human origin was produced (Example 10).The mAb, termed 7B11, is an antibody antagonist of C—C chemokinereceptor 3 of human origin and the functions of the receptor. The 7B11hybridoma cell line was deposited on Sep. 25, 1996 under the terms ofthe Budapest Treaty at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under Accession NumberHB-12195.

Genomic and cDNA clones were also expressed in a variety of systems.Antibody was used to detect expression of receptor from the genomicclone on transfected mammalian cells and baculovirus-transfected insectcells. Stable transfectants of mammalian cells expressing CKR-3 wereconstructed, and the encoded receptor was shown to bind radiolabeledeotaxin specifically and with high affinity, comparable to the bindingaffinity observed with eosinophils. Studies with transfected mammaliancells indicated that the receptor also binds RANTES and MCP-3specifically and with high affinity, but not other CC or CXC chemokinestested. Consistent with the binding data, as shown herein, receptortransfectants generated in a murine B cell lymphoma line migrated inchemotaxis assays in response to eotaxin, RANTES, and MCP-3, but not toany other chemokines tested. When expressed in several heterologoussystems, the human receptor did not significantly bind to MIP-1α underthe conditions used. Moreover, chemotaxis and ligand binding assaysusing eosinophils indicate that RANTES and MCP-3 bind eosinophilsthrough a receptor, which is distinct from C—C chemokine receptor 1, theMIP 1α/RANTES receptor.

The role of MIP-1α as an eosinophil chemoattractant has beencontroversial. Some investigators detect chemotactic responses (Rot, A.,et al., J. Exp. Med., 176: 1489-1495 (1995)), whereas others do not(FIG. 4, Example 1; Ebisawa, M., et al., J. Immunol., 153: 2153-2160(1994); and Ponath, P. D., et al., J. Clin. Invest., (1996)(in press)).Interestingly, MIP-1α is an eosinophil chemoattractant in the mouse, andthis appears to be mediated through the murine CKR-3 homologue, whichalso binds and signals with murine eotaxin (Post, T. W., et al., J.Immunol., 155: 5299-5305 (1995); the teachings of which are incorporatedherein by reference in their entirety).

Using the proteins and antibodies of the present invention, additionalligands, as well as additional cell types (e.g., leukocytes, such asbasophils) which express CKR-3 receptor, can be identified. For example,as described herein, using 7B11, it has been demonstrated that basophilsexpress CCR3. The ability of other chemokines to bind mammalian CKR-3receptors can be assessed according to the present invention.

The cloning and characterization of clones encoding a novel receptor,and the isolation and characterization of the novel CKR-3 receptor whichdemonstrably binds and mediates chemotaxis in response to chemokinessuch as eotaxin, RANTES and MCP-3, suggests that this receptor is amember of a family of seven transmembrane spanning G protein-coupledreceptors which are involved in selective leukocyte chemotaxis andactivation in response to chemokines. The CKR-3 or CCR3 receptor and itsmammalian homologs are distinct from the MIP-1α/RANTES receptor and theMCP-1 receptor (i.e., are receptors other than C—C chemokine receptor 1(CKR-1) and MCP-1 receptor (CKR-2) and their homologs).

Because of the role of chemokine receptors in the selective induction ofleukocyte chemotaxis and leukocyte activation in response tochemoattractants, chemokine receptors play a fundamental role inleukocyte migration, and particularly in migration associated withinflammation. Chemokines, produced at sites of inflammation andinfection, specifically recruit selected leukocyte subtypes from thecirculation to the site of inflammation in the tissues. Subsequent tochemokine binding to a leukocyte chemokine receptor, integrin activationoccurs, and leukocytes adhere firmly to the endothelial cell wall vialeukocyte integrins and endothelial cell adhesion molecules. Theleukocytes become flat in shape, and migrate through the endotheliumtowards sites of inflammation in the tissues. The specificity of aleukocyte for a tissue or inflammatory site is, in many cases,determined at the level of the chemokine-receptor interaction, ratherthan at the level of the adhesion interaction between integrin andcellular adhesion molecules.

RANTES and MCP-3 are among the most potent chemotactic cytokines foreosinophils and basophils. In addition, RANTES is reported to be achemoattractant for memory T cells, a subpopulation of T lymphocytes. Asshown herein, RANTES and MCP-3 can induce chemotaxis of eosinophils.CKR-3 receptor proteins described herein also bind RANTES and MCP-3 withhigh affinity.

As is further shown herein, CKR-3 binds eotaxin specifically and withhigh affinity (comparable to the binding affinity observed witheosinophils), and the CKR-3 receptor is highly restricted in itsexpression. Although a number of chemoattractants have been identifiedfor eosinophils, such as RANTES and MCP-3 (Baggiolini, M. and Dahinden,C. A., Immunol. Today, 15:127-33 (1994); Dahinden, C. A., et al., J.Exp. Med., 179: 751-756 (1994); Kameyoshi, Y, et al., J. Exp. Med., 176:587-592 (1992); Rot, A., et al., J. Exp. Med., 176: 1489-1495 (1995)),as well as PAF, C5a, and IL-16 (Wardlaw, A. J., et al., J. Clin.Invest., 78: 1701-1706 (1986); Gerard, N. P., et al., J. Biol. Chem.,264: 1760-1765 (1989); Rand, T. H., et al., J. Exp. Med., 173: 1521-1528(1991)), these chemoattracants also induce the migration of otherleukocyte cell types. In contrast, the chemokine eotaxin, a potenteosinophil chemoattractant originally identified in guinea pigs andsubsequently in mouse and human, is selectively chemotactic foreosinophils (Jose, P. J., et al., Biochem. Biophys. Res. Commun., 205:788-794 (1994); Jose, P. J., et al., J. Exp. Med., 179: 881-887 (1994);Rothenburg, M. E. et al., Proc. Natl. Acad. Sci. U.S.A., 92: 8960-8964(1995); Ponath, P. D., et al., J. Clin. Invest., 97(3):604-612 (1996)).In addition, eotaxin binds to and signals through CKR-3 with a highdegree of fidelity, in contrast to chemokines such as MCP-3, which bindsCKR-1 and CKR-2 (Ben-Baruch, A., et al., J. Biol. Chem., 270:22123-22128 (1995)) in addition to CKR-3, or MIP-1α, which binds CKR-1and CKR-4 (Neote, K., et al., Cell, 72: 415-425 (1993); Power, C. A., etal., J. Biol. Chem., 270: 19495-19500 (1995)). The restricted expressionof CKR-3 on eosinophils, and the fidelity of eotaxin binding to CKR-3,provides a potential mechanism for the selective recruitment andmigration of eosinophils within tissues. In this regard, the productionof eotaxin within a tissue can lead to selective eosinophil recruitment;eotaxin injection into the skin of rhesus monkeys leads to selectiveeosinophil migration. In addition, eotaxin was shown to recruiteosinophils in vivo at a 10-fold lower dose than RANTES, similar to thein vitro chemotaxis of CKR-3 transfectants (Ponath, P. D., et al., J.Clin. Invest., 97(3):604-612 (1996)).

Modulation of mammalian CKR-3 receptor function according to the presentinvention, through the inhibition or promotion of receptor function,such as binding, signalling or stimulation of a cellular response,provides an effective and selective way of inhibiting or promotingleukocyte-mediated inflammatory action, particularly that ofeosinophils, basophils, and/or T cells. Ligands, inhibitors andpromoters of CKR-3 receptor function, such as those identified asdescribed herein, can be used to modulate leukocyte function fortherapeutic purposes.

Eosinophils do not express the MIP-1α receptor, and do not expresssignificant amounts of MCP-1 receptor. In addition, as noted above,eotaxin and RANTES are some of the most potent chemoattractants foreosinophils, and eotaxin and RANTES bind specifically and with highaffinity to the CKR-3 receptor. As a major eosinophil and lymphocytechemokine receptor, the CKR-3 receptor is an important target forinterfering with or promoting eosinophil, basophil, and/or T lymphocytefunction. Compounds which inhibit or promote CKR-3 receptor function,such as ligands, inhibitors and promoters identified according to thepresent method, are particularly useful for modulating eosinophil,basophil, and/or T cell function for therapeutic purposes.

For example, as described herein, anti-CCR3 antibody 7B11, inhibitseosinophil degranulation induced by binding of eotaxin to CCR-3 (Exmaple11). As also demonstrated herein, 7B 11 inhibits basophil chemotaxis toeotaxin and MCP-4, as well as histamine release by basophils in responseto chemokines (Example 13). Chemokine receptor other names ligandsdefined to date CCR1 CC CKR1 MIP-1α, RANTES, MCP-3 CCR2a, b MCP-1Ra, bMCP-1, MCP-3, MCP-4 CCR3 CKR-3 eotaxin, RANTES, MCP-2, 3, 4 CCR4 RANTES,MIP-1α, MCP-1 CCR5 CC CKR5 RANTES, MIP-1α, MIP-1β CXCR1 IL-8 RA, IL-8 R1IL-8 CXCR2 IL-8 RB, IL-8 R2 IL-8, GROα, NAP-2, ENA-78 CXCR3 none IP-10,Mig CXCR4 Fusin/humstr/Lestr SDF-1Nucleic Acids, Constructs and Vectors

The present invention relates to isolated and/or recombinant (including,e.g., essentially pure) nucleic acids having sequences which encode amammalian (e.g., human) receptor protein designated Eos L2 or C—CChemokine Receptor 3 (CKR-3, also referred to herein as CCR3) or aportion of said receptor. In one embodiment, the nucleic acid or portionthereof encodes a protein or polypeptide having at least one functioncharacteristic of a mammalian C—C chemokine receptor (e.g., a mammalianCKR-3 receptor), such as a binding activity (e.g., ligand, inhibitorand/or promoter binding), a signalling activity (e.g., activation of amammalian G protein, induction of rapid and transient increase in theconcentration of cytosolic free calcium [Ca²⁺]_(i)), and/or stimulationof a cellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes, integrin activation). Thepresent invention also relates more specifically to isolated and/orrecombinant nucleic acids or a portion thereof comprising sequenceswhich encode a mammalian CKR-3 receptor or a portion thereof.

The invention further relates to isolated and/or recombinant nucleicacids that are characterized by (1) their ability to hybridize to: (a) anucleic acid having the sequence SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5, (b) a the complement of any one of SEQ ID NOS:1, 3 or 5, (c) aportion of the foregoing comprising the coding region (nucleotides181-1245 of SEQ ID NO:1, nucleotides 92-1156 of SEQ ID NO:3, ornucleotides 15-1079 of SEQ ID NO:5), or the RNA counterpart of any oneof the foregoing, wherein U is substituted for T; or (2) by theirability to encode a polypeptide having the amino acid sequence SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6 or a functional equivalents thereof(i.e., a polypeptide having ligand binding activity for one or morenatural or physiological ligand(s) of the receptor and/or stimulatoryfunction responsive to ligand binding, such that it can stimulate acellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes); or (3) by bothcharacteristics.

In one embodiment, the percent amino acid sequence identity between SEQID NOS:2, 4 or 6 and functional equivalents thereof is at least about70% (≧70%). In a preferred embodiment, functional equivalents of SEQ IDNOS:2, 4 or 6 share at least about 80% sequence identity with SEQ iIDNOS:2, 4 or 6, respectively. More preferably, the percent amino acidsequence identity between SEQ ID NOS:2, 4 or 6 and functionalequivalents thereof is at least about 90%, and still more preferably, atleast about 95%. Isolated and/or recombinant nucleic acids meeting thesecriteria comprise nucleic acids having sequences identical to sequencesof naturally occurring mammalian CKR-3 receptors and portions thereof,or variants of the naturally occurring sequences. Such variants includemutants differing by the addition, deletion or substitution of one ormore residues, modified nucleic acids in which one or more residues ismodified (e.g., DNA or RNA analogs), and mutants comprising one or moremodified residues.

Such nucleic acids can be detected and isolated by hybridization underhigh stringency conditions or moderate stringency conditions, forexample. “High stringency conditions” and “moderate stringencyconditions” for nucleic acid hybridizations are explained on pages2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1,Suppl. 26, 1991), the teachings of which are incorporated herein byreference (see also Example 2). Factors such as probe length, basecomposition, percent mismatch between the hybridizing sequences,temperature and ionic strength influence the stability of nucleic acidhybrids. Thus, high or moderate stringency conditions can be determinedempirically, depending in part upon the characteristics of the known DNAto which other unknown nucleic acids are being compared for homology.

Isolated and/or recombinant nucleic acids that are characterized bytheir ability to hybridize to a nucleic acid having the sequence SEQ IDNOS: 1, 3 or 5 or the complements of any one of SEQ ID NOS: 1, 3 or 5(e.g. under high or moderate stringency conditions) may further encode aprotein or polypeptide having at least one function characteristic of amammalian C—C chemokine receptor (e.g., a mammalian CKR-3 receptor),such as a binding activity (e.g., ligand, inhibitor and/or promoterbinding), a signalling activity (e.g., activation of a mammalian Gprotein, induction of rapid and transient increase in the concentrationof cytosolic free calcium [Ca²⁺]_(i)), and/or stimulation of a cellularresponse (e.g., stimulation of chemotaxis, exocytosis or inflammatorymediator release by leukocytes, integrin activation).

The signalling function of a protein or polypeptide encoded byhybridizing nucleic acid can be detected by enzymatic assays for Gprotein activity responsive to receptor binding (e.g., exchange of GTPfor GDP on the G protein α subunit, using membrane fractions). G proteincoupling can be further assessed, for example, using assays in whichstimulation by G protein is blocked by treatment or pre-treatment ofcells or a suitable cellular fraction (e.g., membranes) with specificinhibitors of G proteins, such as Bordetella pertussis toxin (Bischoff,S. C. et al., Eur. J. Immunol. 23: 761-767 (1993); Sozzani, S. et al.,J. Immunol. 147: 2215-2221 (1991)).

The stimulatory function of a protein or polypeptide encoded byhybridizing nucleic acid can be detected by standard assays forchemotaxis or mediator release, using cells expressing the protein orpolypeptide (e.g., assays which monitor chemotaxis, exocytosis (e.g., ofenzymes such as eosinophil peroxidase, β-glucuronidase) or mediatorrelease in response to a ligand (e.g., a chemokine such as eotaxin,RANTES or MCP-3) or a promoter.

The binding function of a protein or polypeptide encoded by hybridizingnucleic acid can be detected in binding or binding inhibition assaysusing membrane fractions containing receptor or cells expressingreceptor, for instance (see e.g., Example 9; Van Riper et al., J. Exp.Med., 177: 851-856 (1993); Sledziewski et al., U.S. Pat. No. 5,284,746(Feb. 8, 1994)). Thus, the ability of the encoded protein or polypeptideto bind a ligand, such as eotaxin, RANTES or MCP-3, an inhibitor and/orpromoter, can be assessed. Functions characteristic of a mammalian CKR-3receptor may also be assessed by other suitable methods (see below).

These methods, alone or in combination with other suitable methods canalso be used in procedures for the identification and/or isolation ofnucleic acids which encode a polypeptide having the amino acid sequenceSEQ ID NO: 2, 4, 6 or functional equivalents thereof, and having anactivity detected by the assay. Portions of the isolated nucleic acidswhich encode polypeptide portions of SEQ ID NO: 2, 4 or 6 having acertain function can be also identified and isolated in this manner.

Nucleic acids of the present invention can be used in the production ofproteins or polypeptides. For example, a nucleic acid containing all orpart of the coding sequence for a mammalian CKR-3 receptor, or DNA whichhybridizes to the sequence SEQ ID NO: 1, 3 or 5, or the complement ofany one of SEQ ID NO: 1, 3 or 5, can be incorporated into variousconstructs and vectors created for further manipulation of sequences orfor production of the encoded polypeptide in suitable host cells.

Nucleic acids referred to herein as “isolated” are nucleic acidsseparated away from the nucleic acids of the genomic DNA or cellular RNAof their source of origin (e.g., as it exists in cells or in a mixtureof nucleic acids such as a library), and may have undergone furtherprocessing. “Isolated” nucleic acids include nucleic acids obtained bymethods described herein, similar methods or other suitable methods,including essentially pure nucleic acids, nucleic acids produced bychemical synthesis, by combinations of biological and chemical methods,and recombinant nucleic acids which are isolated. Nucleic acids referredto herein as “recombinant” are nucleic acids which have been produced byrecombinant DNA methodology, including those nucleic acids that aregenerated by procedures which rely upon a method of artificialrecombination, such as the polymerase chain reaction (PCR) and/orcloning into a vector using restriction enzymes. “Recombinant” nucleicacids are also those that result from recombination events that occurthrough the natural mechanisms of cells, but are selected for after theintroduction to the cells of nucleic acids designed to allow and makeprobable a desired recombination event.

Antisense Constructs

In another embodiment, the nucleic acid is an antisense nucleic acid,which is complementary, in whole or in part, to a target moleculecomprising a sense strand, and can hybridize with the target molecule.The target can be DNA, or its RNA counterpart (i.e., wherein T residuesof the DNA are U residues in the RNA counterpart). When introduced intoa cell using methods known in the art or other suitable methods,antisense nucleic acid can inhibit the expression of the gene encoded bythe sense strand. Antisense nucleic acids can be produced by standardtechniques.

In one embodiment, the antisense nucleic acid is wholly or partiallycomplementary to and can hybridize with a target nucleic acid, whereinthe target nucleic acid can hybridize to a nucleic acid having thesequence of the complement of SEQ ID NO:1, 3 or 5. For example,antisense nucleic acid can be complementary to a target nucleic acidhaving the sequence of SEQ ID NO: 5 or a portion thereof sufficient toallow hybridization. In another embodiment, the antisense nucleic acidis wholly or partially complementary to and can hybridize with a targetnucleic acid which encodes a mammalian CKR-3 receptor (e.g., human EosL2 receptor).

Antisense nucleic acids are useful for a variety of purposes, includingresearch and therapeutic applications. For example, a constructcomprising an antisense nucleic acid can be introduced into a suitablecell to inhibit receptor expression. Such a cell provides a valuablecontrol cell, for instance in assessing the specificity ofreceptor-ligand interaction with the parent cell or other related celltypes. In another aspect, such a construct is introduced into some orall of the cells of a mammal. The antisense nucleic acid inhibitsreceptor expression, and inflammatory processes mediated by CKR-3receptors in the cells containing the construct can be inhibited. Thus,an inflammatory disease or condition can be treated using an antisensenucleic acid of the present invention. Suitable laboratory animalscomprising an antisense construct can also provide useful models fordeficiencies of leukocyte function, and of eosinophil deficiency inparticular, and provide further information regarding CKR-3 receptorfunction. Such animals can provide valuable models of infectiousdisease, useful for elucidating the role of leukocytes, such aseosinophils and/or T lymphocytes, in host defenses.

Mammalian Nucleic Acids

Because advances in the understanding and treatment of humaninflammatory and autoimmune diseases and of parasitic infections wouldbe of tremendous benefit, human CKR-3 or CCR3 was the species selectedfor most of the experimental work described herein. However, theapproaches described to isolate and manipulate the genomic and cDNAs ofhuman CKR-3 (Eos L2), to construct vectors and host strains, and toproduce and use the receptor or fragments thereof, can be applied toother mammalian species, including, but not limited to primate (e.g., aprimate other than a human, such as a monkey (e.g., cynomolgus monkey)),bovine (e.g., cows), ovine (e.g., sheep), equine (e.g., horses), canine(e.g., dog), feline (e.g., domestic cat) and rodent (e.g., guinea pig,murine species such as rat, mouse) species. The human CKR-3 cDNA orgenomic clones described here, or sufficient portions thereof, whetherisolated and/or recombinant or synthetic, including fragments within thecoding sequence produced by PCR, can be used as probes to detect and/orrecover homologous CKR-3 genes (homologs) or other related receptorgenes (e.g., novel C—C chemokine receptor genes) from other mammalianspecies (e.g., by hybridization, PCR or other suitable techniques). Thiscan be achieved using the procedures described herein or other suitablemethods.

Proteins And Peptides

The invention also relates to proteins or polypeptides encoded bynucleic acids of the present invention. The proteins and polypeptides ofthe present invention can be isolated and/or recombinant. Proteins orpolypeptides referred to herein as “isolated” are proteins orpolypeptides purified to a state beyond that in which they exist inmammalian cells. “Isolated” proteins or polypeptides include proteins orpolypeptides obtained by methods described herein, similar methods orother suitable methods, including essentially pure proteins orpolypeptides, proteins or polypeptides produced by chemical synthesis,or by combinations of biological and chemical methods, and recombinantproteins or polypeptides which are isolated. Proteins or polypeptidesreferred to herein as “recombinant” are proteins or polypeptidesproduced by the expression of recombinant nucleic acids.

In a preferred embodiment, the protein or polypeptide has at least onefunction characteristic of a mammalian CKR-3 receptor, such as a bindingactivity (e.g., ligand, inhibitor and/or promoter binding), a signallingactivity (e.g., activation of a mammalian G protein, induction of rapidand transient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i)), and/or stimulation of a cellular response (e.g.,stimulation of chemotaxis, exocytosis or inflammatory mediator releaseby leukocytes, integrin activation). As such, these proteins arereferred to as CKR-3 proteins of mammalian origin or mammalian chemokinereceptor 3 proteins, and include, for example, naturally occurringmammalian CKR-3 receptors, variants of those proteins and/or portionsthereof. Such variants include polymorphic variants and natural orartificial mutants, differing by the addition, deletion or substitutionof one or more amino acid residues, or modified polypeptides in whichone or more residues is modified, and mutants comprising one or moremodified residues. An example would be a mammalian CKR-3 receptorprotein which binds eotaxin.

In a particularly preferred embodiment, like naturally occurringmammalian CKR-3 receptor proteins or polypeptides, the mammalian CKR-3receptors of the present invention have ligand binding function for oneor more natural or physiological ligand(s) and/or stimulatory functionresponsive to ligand binding, such that they can stimulate a cellularresponse (e.g., stimulation of chemotaxis, exocytosis or inflammatorymediator release by leukocytes). For example, in the case of a humanchemokine receptor 3 protein, an isolated human CKR-3 protein will bindthe one or more natural or physiological ligand(s). As shown herein, anisolated human CKR-3 protein binds eotaxin and RANTES specifically andwith high affinity, and specifically binds MCP-3. In one embodiment, ahuman CKR-3 receptor protein or polypeptide also triggers chemotaxis,exocytosis or inflammatory mediator release by leukocytes in response toligand binding.

The invention further relates to fusion proteins, comprising a mammalianCKR-3 receptor protein or polypeptide (as described above) as a firstmoiety, linked to a second moiety not occurring in the mammalian CKR-3receptor as found in nature. Thus, the second moiety can be an aminoacid or polypeptide. The first moiety can be in an N-terminal location,C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises a human CKR-3 receptor as thefirst moiety, and a second moiety comprising a linker sequence andaffinity ligand (e.g., an enzyme, an antigen, epitope tag).

Fusion proteins can be produced by a variety of methods. For example,some embodiments can be produced by the insertion of a CKR-3 gene orportion thereof into a suitable expression vector, such asBluescript®7II SK ± (Stratagene), pGEX-4T-2 (Pharmacia) and pET-15b(Novagen). The resulting construct is then introduced into a suitablehost cell for expression. Upon expression, fusion protein can beisolated or purified from a cell lysate by means of a suitable affinitymatrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F. M.et al., eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)). In addition,affinity labels provide a means of detecting CKR-3 receptor proteins orpolypeptides present in a fusion protein. For example, the cell surfaceexpression or presence in a particular cell fraction of a fusion proteincomprising an antigen or epitope affinity label can be detected by meansof an appropriate antibody (see, e.g., Example 3).

The invention also relates to isolated and/or recombinant portions of aCKR-3 receptor of mammalian origin, such as a fragment of a human CKR-3receptor. As is described in more detail below, portions of a mammalianCKR-3 receptor can be produced (e.g., synthetic peptides) and used toproduce antibodies. In one embodiment, an isolated and/or recombinantportion (e.g., a peptide) of a selected mammalian CKR-3 receptor has atleast one immunological property. As used herein, with reference to aportion of a receptor, an immunological property includesimmunoreactivity (bound by antibodies raised against a mammalian CKR-3receptor protein of the present invention, including a portion thereof),immunogenicity (induces an antibody response against itself when used ina suitable immunization protocol), and/or cross-reactivity (inducesantibodies reactive with a selected mammalian receptor). Furthermore,portions of a CKR-3 receptor having at least one function characteristicof mammalian CKR-3 receptors, such as binding activity, signallingactivity, or stimulatory function (stimulation of a cellular response),can also be produced. Extensive studies on the structure and function ofmammalian G protein-coupled receptors provide the basis for being ableto divide mammalian CKR-3 receptors into functional domains (see e.g.,Lefkowitz et al., J. Biol. Chem., 263: 4993-4996 (2988); Panayotou andWaterfield, Curr. Opinion Cell Biol., 1: 167-176 (1989)). Furthermore,portions of the receptor can be produced which have full or partialfunction on their own, or which when joined with another portion of asecond receptor (though fully, partially, or nonfunctional alone),constitute a functional protein having at least one functioncharacteristic of a mammalian CKR-3 receptor (e.g., ligand, inhibitor-or promoter-binding function). (See, e.g., Sledziewski et al., U.S. Pat.No. 5,284,746 regarding the construction and use of hybrid Gprotein-coupled receptors useful in detecting the presence of ligand ina test sample).

Method Of Producing Recombinant Mammalian CKR-3 Receptors

Another aspect of the invention relates to a method of producing amammalian CKR-3 receptor or a portion thereof. Constructs suitable forthe expression of a mammalian CKR-3 receptor or a portion thereof arealso provided. The constructs can be introduced into a suitable hostcell. Cells expressing a recombinant mammalian CKR-3 receptor or aportion thereof can be isolated and maintained in culture. Such cellsare useful for a variety of purposes such as the production of proteinfor characterization, isolation and/or purification, and in bindingassays for the detection of ligands, or inhibitors or promoters ofligand binding. Suitable host cells can be procaryotic, includingbacterial cells such as E. coli, B. subtilis and or other suitablebacteria, or eucaryotic, such as fungal or yeast cells (e.g., Pichiapastoris, Aspergillus species, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Neurospora crassa), or other lower eucaryoticcells, and cells of higher eucaryotes such as those from insects (e.g.,Sf9 insect cells) or mammals (e.g., 293 cells, Chinese hamster ovarycells (CHO)). (See, e.g., Ausubel, F. M. et al., eds. Current Protocolsin Molecular Biology, Greene Publishing Associates and John Wiley & SonsInc., (1993)).

Host cells which produce a recombinant mammalian CKR-3 receptor protein,portion thereof, or fusion protein can be produced as follows. A nucleicacid encoding all or part of the coding sequence for a mammalian CKR-3receptor or fusion protein can be inserted into a nucleic acid vector,e.g., a DNA vector, such as a plasmid, virus or other suitable repliconfor expression. A variety of vectors are available, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

The transcriptional and/or translational signals of a selected CKR-3receptor can be used to direct expression. Alternatively, suitableexpression vectors are available. Suitable vectors for expression of anucleic acid encoding all or part of the coding sequence for a mammalianCKR-3 receptor or fusion protein can contain a number of additionalcomponents, including, but not limited to one or more of the following:an origin of replication; a selectable marker gene; one or moreexpression control elements, such as a transcriptional control element(e.g., a promoter, an enhancer, terminator), and/or one or moretranslation signals; a signal sequence or leader sequence (for membranetargeting encoded e.g., by the vector, receptor or other source).

A promoter can be provided for expression in a suitable host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding the receptor protein,portion thereof or fusion protein, such that it is capable of directingexpression of the encoded polypeptide. A variety of suitable promotersfor procaryotic (e.g., lac, tac, T3, T7 promoters for E. coli) andeukaryotic (e.g., yeast alcohol dehydrogenase (ADH1), SV40, CMV) hostsare available.

In addition, the expression vectors typically comprise a selectablemarker for selection of host cells carrying the vector and an origin orreplication, in the case of replicable expression vector. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in prokaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeukaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. The present invention alsorelates to cells carrying these expression vectors.

When the nucleic acid encoding the receptor protein or polypeptide isinserted into the vector, operably linked to one or more of thesecomponents, and the resulting construct is introduced into host cellsmaintained under conditions suitable for expression, the receptorprotein or polypeptide is produced. The construct can be introduced intocells by a method appropriate to the host cell selected (e.g.,transformation, transfection, electroporation, infection). Forproduction of receptor, host cells comprising the construct aremaintained under conditions appropriate for expression, e.g., in thepresence of inducer (e.g., n-butyrate), suitable media supplemented withappropriate salts, growth factors, antibiotic, nutritional supplements,etc.

Antibodies

The invention further relates to antibodies reactive with a CKR-3receptor or portion thereof. In one embodiment, antibodies are raisedagainst an isolated and/or recombinant mammalian CKR-3 protein includingportions thereof (e.g., a peptide). In a preferred embodiment, theantibodies specifically bind CKR-3 (CCR3) receptor(s) or a portionthereof. Antibodies which can inhibit one or more functionscharacteristic of a mammalian CKR-3 (CCR3), such as a binding activity,a signalling activity, and/or stimulation of a cellular response arealso encompassed by the present invention, such as an antibody which caninhibit binding of a ligand (i.e., one or more ligands) to CKR-3 (CCR3)and/or one or more functions mediated by CKR-3 (CCR3) in response to aligand. For example, monoclonal antibody 7B11 can inhibit binding ofeotaxin, RANTES, MCP-2, MCP-3 and MCP-4 to human CKR-3 (CCR3).Furthermore, 7B11 can inhibit functions mediated by human CKR-3 (CCR3),including chemokine-induced calcium flux, eosinophil and basophilchemotaxis, histamine release and release of other granule components.

In a particularly preferred embodiment, the antibodies of the presentinvention have specificity for human CKR-3 (CCR3), and have an epitopicspecificity similar to that of murine 7B11 monoclonal antibody describedherein. Antibodies with an epitopic specificity similar to that ofmurine 7B 11 monoclonal antibody can be identified by their ability tocompete with murine 7B11 for binding to human CCR3 (e.g., to cellsbearing human CCR3, such as eosinophils, basophils, or cells transfectedwith a nucleic acid of the present invention), for example.

The antibodies of the present invention can be polyclonal or monoclonal(see e.g., Example 5), and the term antibody is intended to encompassboth polyclonal and monoclonal antibodies. Antibodies of the presentinvention can be raised against an appropriate immunogen, includingproteins or polypeptides of the present invention, such as isolatedand/or recombinant mammalian CKR-3 receptor protein or portion thereof,or synthetic molecules, such as synthetic peptides. In addition, cellswhich express receptor, such as transfected cells, can be used asimmunogens or in a screen for antibody which binds receptor. See forexample, Chuntharapai et al., J. Immunol. 152: 1783-1789 (1994)).

Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)). Generally, a hybridoma can be produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, are obtained from animals immunizedwith the antigen of interest. The fused cells (hybridomas) can beisolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, comprising portions derived from different species,are also encompassed by the present invention and the term “antibody”.The various portions of these antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; andWinter, European Patent No. 0,239,400 B1. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

In addition, functional fragments of antibodies, including fragments ofchimeric, humanized, primatized or single chain antibodies, can also beproduced. Functional fragments of foregoing antibodies retain at leastone binding function and/or modulation function of the full-lengthantibody from which they are derived. Preferred functional fragmentsretain an antigen binding function of a corresponding full lengthantibody (e.g., specificity for a mammalian CKR-3 (CCR3)). Particularlypreferred functional fragments retain the ability to inhibit one or morefunctions characteristic of a mammalian CKR-3 (CCR3), such as a bindingactivity, a signalling activity, and/or stimulation of a cellularresponse. For example, in one embodiment, a functional fragment caninhibit the interaction of CKR-3 (CCR3) with one or more of its ligands(e.g., eotaxin, RANTES, MCP-2, MCP-3, MCP-4) and/or can inhibit one ormore receptor-mediated functions, such as eosinophil or basophilchemotaxis and/or degranulation induced by chemokine binding to CKR-3(CCR3). For example, antibody fragments capable of binding to amammalian CKR-3 receptor or portion thereof, including, but not limitedto, Fv, Fab, Fab′ and F(ab′)₂ fragments are encompassed by theinvention. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)₂ heavy chainportion can be designed to include DNA sequences encoding the CH1 domainand hinge region of the heavy chain.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. Accordingly,the present invention relates to a humanized immunoglobulin havingbinding specificity for a mammalian CCR3 (e.g., human CCR3), saidimmunoglobulin comprising an antigen binding region of nonhuman origin(e.g., rodent) and at least a portion of an immunoglobulin of humanorigin (e.g., a human framework region, a human constant region orportion thereof). For example, the humanized antibody can compriseportions derived from an immunoglobulin of nonhuman origin with therequisite specificity, such as a mouse, and from immunoglobulinsequences of human origin (e.g., chimeric immunoglobulin), joinedtogether chemically by conventional techniques (e.g., synthetic) orprepared as a contiguous polypeptide using genetic engineeringtechniques (e.g., DNA encoding the protein portions of the chimericantibody can be expressed to produce a contiguous polypeptide chain).Another example of a humanized immunoglobulin of the present inventionis an immunoglobulin containing one or more immunoglobulin chainscomprising a CDR of nonhuman origin (e.g., one or more CDRs derived froman antibody of nonhuman origin) and a framework region derived from alight and/or heavy chain of human origin (e.g., CDR-grafted antibodieswith or without framework changes). In one embodiment, the humanizedimmunoglobulin can compete with murine 7B11 monoclonal antibody forbinding to human CCR3 (e.g., to cells bearing human CCR3, such aseosinophils, basophils, or cells transfected with a nucleic acid of thepresent invention). In a preferred embodiment, the antigen bindingregion of the humanized immunoglobulin is derived from 7B11 monoclonalantibody. Chimeric or CDR-grafted single chain antibodies are alsoencompassed by the term humanized immunoglobulin. See, e.g., Cabilly etal., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.0,125,023 B1; Queen et al., European Patent No. 0,451,216 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; Padlan, E. A. et al., EuropeanPatent Application No. 0,519,596 A1. See also, Ladner et al., U.S. Pat.No. 4,946,778; Huston, U.S. Pat. No. 5,476,786; and Bird, R. E. et al.,Science, 242: 423-426 (1988)), regarding single chain antibodies.

The antibodies of the present invention are useful in a variety ofapplications, including research, diagnostic and therapeuticapplications. In one embodiment, the antibodies are labeled with asuitable label (e.g., fluorescent label, chemiluminescent label, isotopelabel, epitope or enzyme label). For instance, they can be used toisolate and/or purify receptor or portions thereof, and to studyreceptor structure (e.g., conformation) and function.

The antibodies of the present invention can also be used to modulatereceptor function in research and therapeutic applications. Forinstance, antibodies can act as inhibitors to inhibit (reduce orprevent) (a) binding (e.g., of a ligand, a second inhibitor or apromoter) to the receptor, (b) a receptor signalling, (c) and/or astimulatory function. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change). For example, antibodies caninhibit receptor function by inhibiting binding of a ligand, or bydesensitization (with or without inhibition of binding of a ligand).

Antibodies which bind receptor can also act as agonists of receptorfunction, triggering or stimulating a receptor function, such as asignalling and/or a stimulatory function of a receptor (e.g.,chemotaxis, exocytosis or pro-inflammatory mediator release) uponbinding to receptor.

In addition, the various antibodies of the present invention can be usedto detect or measure the expression of receptor, for example, onleukocytes such as eosinophils, basophils, and lymphocytes, or on cellstransfected with a receptor gene. Thus, they also have utility inapplications such as cell sorting (e.g., flow cytometry, fluorescenceactivated cell sorting), for diagnostic or research purposes.

Anti-idiotypic antibodies are also provided. Anti-idiotypic antibodiesrecognize antigenic determinants associated with the antigen-bindingsite of another antibody. Anti-idiotypic antibodies can be prepared aagainst second antibody by immunizing an animal of the same species, andpreferably of the same strain, as the animal used to produce the secondantibody. See e.g., U.S. Pat. No. 4,699,880.

In one embodiment, antibodies are raised against receptor or a portionthereof, and these antibodies are used in turn to produce ananti-idiotypic antibody. The anti-Id produced thereby can bind compoundswhich bind receptor, such as ligands, inhibitors or promoters ofreceptor function, and can be used in an immunoassay to detect oridentify or quantitate such compounds. Such an anti-idiotypic antibodycan also be an inhibitor of receptor function, although it does not bindreceptor itself.

Anti-idiotypic (i.e., Anti-Id) antibody can itself be used to raise ananti-idiotypic antibody (i.e., Anti-anti-Id). Such an antibody can besimilar or identical in specificity to the original immunizing antibody.In one embodiment, antibody antagonists which block binding to receptorcan be used to raise Anti-Id, and the Anti-Id can be used to raiseAnti-anti-Id, which can have a specificity which is similar or identicalto that of the antibody antagonist. These anti-anti-Id antibodies can beassessed for inhibitory effect on receptor function to determine if theyare antagonists.

Single chain, and chimeric, humanized or primatized (CDR-grafted), aswell as chimeric or CDR-grafted single chain anti-idiotypic antibodiescan be prepared, and are encompassed by the term anti-idiotypicantibody. Antibody fragments of such antibodies can also be prepared.

Identification Of Ligands, Inhibitors Or Promoters Of Receptor Function

As used herein, a ligand is a substance which binds to a receptorprotein. A ligand of a selected mammalian CKR-3 receptor is a substancewhich binds to the selected mammalian receptor. In one embodiment, aligand can bind selectively to two or more mammalian chemokinereceptors, including CKR-3. In a preferred embodiment, ligand binding ofa mammalian CKR-3 receptor occurs with high affinity. The term ligandrefers to substances including, but not limited to, a natural ligand,whether isolated and/or purified, synthetic, and/or recombinant, ahomolog of a natural ligand (e.g., from another mammal), antibodies,portions of such molecules, and other substances which bind receptor. Anatural ligand of a selected mammalian receptor can bind to the receptorunder physiological conditions, and is of a mammalian origin which isthe same as that of the mammalian CKR-3 receptor. The term ligandencompasses substances which are inhibitors or promoters of receptoractivity, as well as substances which bind but lack inhibitor orpromoter activity.

As used herein, an inhibitor is a substance which inhibits at least onefunction characteristic of a mammalian C—C chemokine receptor (e.g., amammalian CKR-3 receptor), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),and/or stimulation of a cellular response. The term inhibitor refers tosubstances including antagonists which bind receptor (e.g., an antibody,a mutant of a natural ligand, other competitive inhibitors of ligandbinding), and substances which inhibit receptor function without bindingthereto (e.g., an anti idiotypic antibody).

As used herein, a promoter is a substance which promotes (induces orenhances) at least one function characteristic of a mammalian C—Cchemokine receptor (e.g., a mammalian CKR-3 receptor), such as a bindingactivity (e.g., ligand, inhibitor and/or promoter binding), a signallingactivity (e.g., activation of a mammalian G protein, induction of rapidand transient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i)), and/or stimulation of a cellular response. The termpromoter refers to substances including agonists which bind receptor(e.g., an antibody, a homolog of a natural ligand from another species),and substances which promote receptor function without binding thereto(e.g., by activating an associated protein).

The assays described below, which rely upon the nucleic acids andproteins of the present invention, can be used, alone or in combinationwith each other or other suitable methods, to identify ligands,inhibitors or promoters of a mammalian CKR-3 receptor protein orpolypeptide. Human CKR-3 does not usually exist in cells at levelssuitable for high-throughput screening; thus, cells which contain andexpress a nucleic acid of the present invention are particularlyvaluable in identifying ligands, inhibitors and promoters of CKR-3receptor proteins.

Upon isolation of a CKR-3 receptor gene from a mammal, the gene can beincorporated into an expression system to produce a receptor protein orpolypeptide as described above. An isolated and/or recombinant receptorprotein or polypeptide, such as a receptor expressed in cells stably ortransiently transfected with a construct comprising a nucleic acid ofthe present invention, or in a cell fraction (e.g., membrane fractionfrom transfected cells) containing receptor, can be used in tests forreceptor function. The receptor can be further purified if desired.Testing of receptor function can be carried out in vitro or in vivo.

An isolated, recombinant mammalian CKR-3 receptor protein, such as ahuman CKR-3 receptor as that shown in FIGS. 1A-1D (see also, SEQ IDNO:2), FIG. 2A-2C (see also, SEQ ID NO:4) or SEQ ID NO:6, can be used inthe present method, in which the effect of a compound is assessed bymonitoring receptor function as described herein or using other suitabletechniques. For example, stable or transient transfectants, such asA31/293/#20 stable transfectants (see e.g., Example 9), stabletranfectants of mouse L1-2 pre-B cells (see e.g., Example 3),baculovirus infected Sf9 cells (see e.g., Example 4), can be used inbinding assays. Stable transfectants of mouse L1-2 pre-B cells or ofother suitable cells capable of chemotaxis can be used (see e.g.,Example 3) in chemotaxis assays, for example.

According to the method of the present invention, compounds can beindividually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography). The presence of one or morecompounds (e.g., a ligand, inhibitor, promoter) in a test sample canalso be determined according to these methods.

Large combinatorial libraries of compounds (e.g., organic compounds,recombinant or synthetic peptides, “peptoids”, nucleic acids) producedby combinatorial chemical synthesis or other methods can be tested (seee.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 2678-2685 (1994) andreferences cited therein; see also, Ohlmeyer, M. H. J. et al., Proc.Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al.,Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to taggedcompounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible.

In one embodiment, phage display methodology is used. For example,receptor is contacted with a phage (e.g., a phage or collection of phagesuch as a library) displaying a polypeptide under conditions appropriatefor receptor binding (e.g., in a suitable binding buffer). Phage boundto receptor is selected using standard techniques or other suitablemethods. Phage can be separated from receptor using a suitable elutionbuffer. For example, a change in the ionic strength or pH can lead to arelease of phage. Alternatively, the elution buffer can comprise arelease component or components designed to disrupt binding of compounds(e.g., one or more compounds which can disrupt binding of the displayedpeptide to the receptor, such as a ligand, inhibitor, and/or promoterwhich competitively inhibits binding). Optionally, the selection processcan be repeated or another selection step can be used to further enrichfor phage which bind receptor. The displayed polypeptide ischaracterized (e.g., by sequencing phage DNA). The polypeptidesidentified can be produced and further tested for ligand binding,inhibitor and/or promoter function. Analogs of such peptides can beproduced which will have increased stability or other desirableproperties.

In one embodiment, phage expressing and displaying a fusion proteinscomprising a coat protein with an N-terminal peptide encoded by randomsequence nucleic acids can be produced. Suitable host cells expressing areceptor protein or polypeptide of the present invention are contactedwith the phage, bound phage are selected, recovered and characterized.(See e.g., Doorbar, J. and G. Winter, J. Mol. Biol., 244: 361 (1994)discussing a phage display procedure used with a G protein-coupledreceptor).

Other sources of potential ligands, inhibitors and/or promoters of amammalian CKR-3 receptor include, but are not limited to, substancessuch as other chemoattractants; other chemokines (e.g., eotaxin), suchas a mammalian chemokine from the same mammal as the receptor, fromanother mammal (e.g., for a human receptor, a homolog of a humanchemokine obtained from a non-human source); variants of otherchemoattractants or chemokines, such as naturally occurring, syntheticor recombinant variants; other mammalian CKR-3 receptor ligands,inhibitors and/or promoters (e.g., antibodies, antagonists, agonists),and variants thereof; other G-protein coupled receptor ligands,inhibitors and/or promoters (e.g., antagonists or agonists); and solubleportions of a mammalian CKR-3 receptor, such as a suitable receptorpeptide or analog which can inhibit receptor function (see e.g., Murphy,R. B., WO 94/05695).

The in vitro method of the present invention can be used inhigh-throughput screening. These assays can be adapted for processinglarge numbers of samples (e.g., a 96 well format). For such screening,use of a host cell expressing receptor, instead of isolated eosinophils,is preferred because of the difficulty in isolating eosinophils.

For binding assays, high level expression of receptor in a suitable hostcell is preferred. Expression of receptor can be monitored in a varietyof ways. For instance, expression can be monitored using antibodies ofthe present invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors; see Example 3).

Binding Assays

The isolated and/or recombinant receptor proteins, portions thereof, orsuitable fusion proteins of the present invention, can be used in amethod to select and identify compounds which bind to a (one or more)mammalian CKR-3 receptor protein, such as human CKR-3 receptor, andwhich are ligands, or potential inhibitors or promoters of receptoractivity. Compounds selected by the method, including ligands,inhibitors or promoters, can be further assessed for an inhibitory orstimulatory effect on receptor function and/or for therapeutic utility.

In one embodiment, compounds which bind to an active, isolated and/orrecombinant mammalian CKR-3 receptor protein or polypeptide areidentified by the method. In this embodiment, the receptor protein orpolypeptide used has at least one function characteristic of a CKR-3receptor, such as a signalling activity (e.g., activation of a mammalianG protein), stimulatory function (e.g., stimulation of chemotaxis orinflammatory mediator release), and/or binding function (e.g., ligand,inhibitor and/or promoter binding). In a particularly preferredembodiment, the isolated and/or recombinant mammalian CKR-3 receptorprotein or polypeptide has ligand binding function, such that it binds anatural ligand of the receptor.

For example, an isolated and/or recombinant mammalian CKR-3 receptorprotein or polypeptide can be maintained under conditions suitable forbinding, the receptor is contacted with a compound to be tested, andbinding is detected or measured. In one embodiment, a receptor proteincan be expressed in cells stably or transiently transfected with aconstruct comprising a nucleic acid sequence which encodes a receptor ofthe present invention. The cells are maintained under conditionsappropriate for expression of receptor. The cells are contacted with acompound under conditions suitable for binding (e.g., in a suitablebinding buffer), and binding is detected by standard techniques. Tomeasure binding, the extent of binding can be determined relative to asuitable control (e.g., compared with background determined in theabsence of compound, compared with binding of a second compound (i.e., astandard), compared with binding of compound to untransfected cells).Optionally, a cellular fraction, such as a membrane fraction, containingreceptor can be used in lieu of whole cells (see e.g., Example 9).

In one embodiment, the compound is labeled with a suitable label (e.g.,fluorescent label, isotope label), and binding is determined bydetection of the label. Specificity of binding can be assessed bycompetition or displacement, for example, using unlabeled compound or asecond ligand as competitor.

Ligands of the mammalian receptor, including natural ligands from thesame mammalian species or from another species, can be identified inthis manner. The binding activity of a promoter or inhibitor which bindsreceptor can also be assessed using such a ligand binding assay.

Binding inhibition assays can also be used to identify ligands, andinhibitors and promoters which bind receptor and inhibit binding ofanother compound such as a ligand. For example, a binding assay can beconducted in which a reduction in the binding of a first compound (inthe absence of a second compound), as compared binding of the firstcompound in the presence of the second compound, is detected ormeasured. The receptor can be contacted with the first and secondcompounds simultaneously, or one after the other, in either order. Areduction in the extent of binding of the first compound in the presenceof the second compound, is indicative of inhibition of binding by thesecond compound. For example, binding of the first compound could bedecreased or abolished.

In one embodiment, direct inhibition of the binding of a first compound(e.g., a chemokine such as RANTES) to a human CKR-3 receptor by a secondtest compound is monitored. For example, the ability of a compound toinhibit the binding of ¹²⁵I-labeled RANTES or ¹²⁵I-labeled MCP-3 tohuman CKR-3 can be monitored. Such an assay can be conducted usingeither whole cells (e.g., eosinophils, or a suitable cell linecontaining nucleic acid encoding a human CKR-3 receptor) or a membranefraction from said cells, for instance.

Other methods of identifying the presence of a compound(s) which bind areceptor are available, such as methods which monitor events which aretriggered by receptor binding, including signalling function and/orstimulation of a cellular response (See below).

It will be understood that the inhibitory effect of antibodies of thepresent invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method in which the first compound in the assay is anotherantibody, under conditions suitable for antibody binding.

Ligands, as well as receptor-binding inhibitors (e.g., antagonists) andpromoters (e.g., agonists), which are identified in this manner, can befurther assessed to determine whether, subsequent to binding, they actto inhibit or activate other functions of CKR-3 receptors and/or toassess their therapeutic utility.

Signalling Assays

The binding of a ligand or promoter, such as an agonist, can result insignalling by a G protein-coupled receptor, and the activity of Gproteins is stimulated. The induction of induce signalling function by acompound can be monitored using any suitable method. For example, Gprotein activity, such as hydrolysis of GTP to GDP, or later signallingevents triggered by receptor binding, such as induction of rapid andtransient increase in the concentration of intracellular (cytosolic)free calcium [Ca²⁺]_(i), can be assayed by methods known in the art orother suitable methods (see e.g., Neote, K. et al., Cell, 72: 415-4251993); Van Riper et al., J. Exp. Med., 177: 851-856 (1993); Dahinden, C.A. et al., J. Exp. Med., 179: 751-756 (1994).

The functional assay of Sledziewski et al. using hybrid G proteincoupled receptors can also be used to monitor the ability a ligand orpromoter to bind receptor and activate a G protein (Sledziewski et al.,U.S. Pat. No. 5,284,746, the teachings of which are incorporated hereinby reference).

A biological response of the host cell (triggered by binding to hybridreceptor) is monitored, detection of the response being indicative ofthe presence of ligand in the test sample. Sledziewski et al. describesa method of detecting the presence of a ligand in a test sample, whereinthe ligand is a compound which is capable of being bound by theligand-binding domain of a receptor. In one embodiment of the method,yeast host cells are transformed with a DNA construct capable ofdirecting the expression of a biologically active hybrid Gprotein-coupled receptor (i.e., a fusion protein). The hybrid receptorcomprises a mammalian G protein-coupled receptor having at least onedomain other than the ligand-binding domain replaced with acorresponding domain of a yeast G protein-coupled receptor, such as aSTE2 gene product. The yeast host cells containing the construct aremaintained under conditions in which the hybrid receptor is expressed,and the cells are contacted with a test sample under conditions suitableto permit binding of ligand to the hybrid receptor. The assay isconducted as described and the biological response of the host cell(triggered by binding to hybrid receptor) is monitored, detection of theresponse being indicative of a signalling function.

For instance, an assay is provided in which binding to a hybrid receptorderived from STE2 gene product leads to induction of the BAR1 promoter.Induction of the promoter is measured by means of a reporter gene(β-gal), which is linked to the BAR1 promoter and introduced into hostcells on a second construct. Expression of the reporter gene can bedetected by an in vitro enzyme assay on cell lysates or by the presenceof blue colonies on plates containing an indicator (X-gal) in themedium, for example.

In another embodiment, the assay is used to identify potentialinhibitors of receptor function. The inhibitory activity of a compoundcan be determined using a ligand or promoter in the assay, and assessingthe ability of the compound to inhibit the activity induced by ligand orpromoter.

Variants of known ligands can also be screened for reduced ability(decreased ability or no ability) to stimulate activity of a coupled Gprotein. In this embodiment, although the compound has ligand bindingactivity (as determined by another method in advance or later),engagement of the receptor does not trigger or only weakly triggersactivity of a coupled G protein. Such compounds are potentialantagonists, and can be further assessed using a suitable assay. Forinstance, the same assay can be conducted in the presence of a ligand orpromoter, and the ability of the compound to inhibit the activity of aligand or promoter is assessed.

Chemotaxis and Assays of Cellular Stimulation

Chemotaxis assays can also be used to assess receptor function. Theseassays are based on the functional migration of cells in vitro or invivo induced by a compound, and can be used to assess the binding and/orchemoattractant effect of ligands, inhibitors, or promoters. The use ofan in vitro transendothelial chemotaxis assay is described in Example 1.Springer et al. describe a transendothelial lymphocyte chemotaxis assay(Springer et al., WO 94/20142, published Sep. 15, 1994, the teachings ofwhich are incorporated herein by reference; see also Berman et al.,Immunol Invest. 17: 625-677 (1988)). Migration across endothelium intocollagen gels has also been described (Kavanaugh et al., J. Immunol,146: 4149-4156 (1991)). Stable transfectants of mouse L1-2 pre-B cellsor of other suitable host cells capable of chemotaxis can be used (seee.g., Example 3) in chemotaxis assays, for example.

Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell (such as a leukocyte (e.g., lymphocyte,eosinophil, basophil)) into or through a barrier (e.g., endothelium, afilter), toward increased levels of a compound, from a first surface ofthe barrier toward an opposite second surface. Membranes or filtersprovide convenient barriers, such that the directional movement ormigration of a suitable cell into or through a filter, toward increasedlevels of a compound, from a first surface of the filter toward anopposite second surface of the filter, is monitored. In some assays, themembrane is coated with a substance to facilitate adhesion, such asICAM-1, fibronectin or collagen.

For example, one can detect or measure the migration of cells in asuitable container (a containing means), from a first chamber into orthrough a microporous membrane into a second chamber which contains acompound to be tested, and which is divided from the first chamber bythe membrane. A suitable membrane, having a suitable pore size formonitoring specific migration in response to compound, including, forexample, nitrocellulose, polycarbonate, is selected. For example, poresizes of about 3-8 microns, and preferably about 5-8 microns can beused. Pore size can be uniform on a filter or within a range of suitablepore sizes.

To assess migration, the distance of migration into the filter, thenumber of cells crossing the filter that remain adherent to the secondsurface of the filter, and/or the number of cells that accumulate in thesecond chamber can be determined using standard techniques (e.g.,microscopy). In one embodiment, the cells are labeled with a detectablelabel (e.g., radioisotope, fluorescent label, antigen or epitope label),and migration can be assessed by determining the presence of the labeladherent to the membrane and/or present in the second chamber using anappropriate method (e.g., by detecting radioactivity, fluorescence,immunoassay). The extent of migration induced by a compound can bedetermined relative to a suitable control (e.g., compared to backgroundmigration determined in the absence of the compound, to the extent ofmigration induced by a second compound (i.e., a standard), compared withmigration of untransfected cells induced by the compound).

Chambers can be formed from various solids, such as plastic, glass,polypropylene, polystyrene, etc. Membranes which are detachable from thechambers, such as a BIOCOAT (Collaborative Biomedical Products) orTRANSWELL (Costar, Cambridge, Mass.) culture insert, facilitate countingadherent cells.

In the container, the filter is situated so as to be in contact withfluid containing cells in the first chamber, and the fluid in the secondchamber. Other than the test compound or additional ligand, inhibitor,or promoter present for the purpose of the assay, the fluid on eitherside of the membrane is preferably the same or substantially similar.The fluid in the chambers can comprise protein solutions (e.g., bovineserum albumin, fetal calf serum, human serum albumin) which may act toincrease stability and inhibit nonspecific binding of cells, and/orculture media.

In a preferred embodiment, particularly for eosinophils, eosinophil-likecells, lymphocytes, or cells expressing a CKR-3 receptor,transendothelial migration is monitored. A transendothelial migrationassay is preferred. Such assays are better physiological models, becausethey more accurately recapitulate in vivo conditions in which leukocytesemigrate from blood vessels toward chemoattractants present in thetissues at sites of inflammation by crossing the endothelial cell layerlining the vessel wall. In addition, transendothelial assays have lowerbackground (signal to noise ratio).

In this embodiment, transmigration through an endothelial cell layerassessed. To prepare the cell layer, endothelial cells can be culturedon a microporous filter or membrane, optionally coated with a substancesuch as collagen, fibronectin, or other extracellular matrix proteins,to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.) or a suitable cell line, such as the ECV 304cell line used in Example 1. To assay chemotaxis in response to aparticular mammalian receptor, endothelial cells of the same mammal arepreferred; however endothelial cells from a heterologous mammalianspecies or genus can also be used.

Generally, the assay is performed by detecting the directional migrationof cells into or through a membrane or filter, in a direction towardincreased levels of a compound, from a first surface of the filtertoward an opposite second surface of the filter, wherein the filtercontains an endothelial cell layer on a first surface. Directionalmigration occurs from the area adjacent to the first surface, into orthrough the membrane, towards a compound situated on the opposite sideof the filter. The concentration of compound present in the areaadjacent to the second surface, is greater than that in the areaadjacent to the first surface.

In one embodiment, a chemotaxis is used to test for ligand or promoteractivity of a compound, a composition comprising cells capable ofmigration and expressing a mammalian CKR-3 receptor are placed in thefirst chamber, and a composition comprising the compound to be tested isplaced in the second chamber, preferably in the absence of other ligandsor promoters capable of inducing chemotaxis of the cells in the firstchamber (having chemoattractant function). However, one or more ligandsor promoters having chemoattractant function may be present. Compoundswhich can bind receptor and induce chemotaxis of the cells expressing amammalian CKR-3 receptor in this assay are ligands or promoters ofreceptor function.

In one embodiment used to test for an inhibitor, a compositioncomprising cells capable of migration and expressing a mammalian CKR-3receptor are placed in the first chamber. A composition comprising oneor more ligands or promoters capable of inducing chemotaxis of the cellsin the first chamber (having chemoattractant function) is placed in thesecond chamber. Either shortly before the cells are placed in the firstchamber, or simultaneously with the cells, a composition comprising thecompound to be tested is placed, preferably, in the first chamber.Compounds which can bind receptor and inhibit the induction ofchemotaxis, by a ligand or promoter, of the cells expressing a mammalianCKR-3 receptor in this assay are inhibitors of receptor function (i.e.,inhibitors of stimulatory function). A reduction in the extent ofmigration induced by the ligand or promoter in the presence of the testcompound, is indicative of inhibitory activity. (see e.g., Example 5).Separate binding studies (see above) could be performed to determinewhether inhibition is a result of binding of the test compound toreceptor or occurs via a different mechanism.

In vivo assays which monitor leukocyte infiltration of a tissue, inresponse to injection of a compound in the tissue, are described below(see Models of Inflammation). These models measure the ability of cellsto respond to a ligand or promoter by emigration and chemotaxis to asite of inflammation.

In addition to the methods described, the effects of a ligand, inhibitoror promoter on the stimulatory function of the receptor can be assessedby monitoring cellular responses induced by active receptor, usingsuitable host cells containing receptor. Similarly, these assays can beused to determine the function of a receptor. For instance, exocytosis(e.g., degranulation of eosinophils leading to release of eosinophilcationic protein and/or one or more enzymes, or other granulecomponents; release of histamine from basophils), inflammatory mediatorrelease (such as release of bioactive lipids such as leukotrienes (e.g.,leukotriene C₄)), and respiratory burst (Rot, A. et al., J. Exp. Med.,176: 1489-1495 (1992)), can be monitored by methods known in the art orother suitable methods. See e.g., Bischoff. S. C. et al., Eur. J.Immunol., 23: 761-767 (1993) and Baggliolini, M. and C. A. Dahinden,Immunology Today, 15: 127-133 (1994) and references cited therein).

In one embodiment, a ligand, inhibitor and/or promoter is identified bymonitoring the release of an enzyme upon degranulation or exocytosis bya cell capable of this function. Cells containing a nucleic acid of thepresent invention, which encodes an active receptor protein capable ofstimulating exocytosis or degranulation are maintained in a suitablemedium under suitable conditions, whereby receptor is expressed anddegranulation can be induced. The receptor is contacted with a compoundto be tested, and enzyme release is assessed. The release of an enzymeinto the medium can be detected or measured using a suitable assay, suchas in an immunological assay, or biochemical assay for enzyme activity.

The medium can be assayed directly, by introducing components of theassay (e.g., substrate, co-factors, antibody) into the medium (e.g.,before, simultaneous with or after the cells and compound are combined).Alternatively, the assay can be performed on medium which has beenseparated from the cells or further fractionated prior to assay.

For example, convenient assays for are available for enzymes such asglucuronidase and eosinophil peroxidase (White, S. R. et al., A kineticassay for eosinophil peroxidase activity in eosinophils and eosinophilconditioned media, J. Immunol. Methods, 144(2): 257-63 (1991)).

Stimulation of degranulation by a compound can be indicative that thecompound is a ligand or promoter of a mammalian CKR-3 receptor. Inanother embodiment, inhibition of degranulation is indicative of aninhibitor. In this embodiment, the cells expressing receptor arecombined with a ligand or promoter, and a compound to be tested is addedbefore, after or simultaneous therewith.

Models of Inflammation

A variety of in vivo models of inflammation are available, which can beused to assess the effects of ligands, inhibitors, or promoters in vivoas therapeutic agents.

For example, primate models with eosinophilic infiltration to the lung,are available for in vivo testing (see e.g., Wegner, C. D. et al.,Science, 247: 456 (1990)). In one embodiment, an antibody (e.g., amonoclonal antibody) which reacts with human CKR-3, and whichcross-reacts with primate CKR-3, is administered to the animal. A numberof parameters can be measured to assess in vivo efficacy including, butnot limited to, the number of eosinophils in broncoalveolar lavagefluid, respiratory compliance, and respiratory rate. A decrease insymptoms of airway hypersensitivity is indicative of therapeuticbenefit.

In addition, a sheep model for asthma, a guinea pig model for passivecutaneous anaphylaxis, or other suitable model can be used to assesscompounds in vivo (see e.g., Weg, V. B. et al., J. Exp. Med., 177: 561(1993); Abraham, W. M. et al., J. Clin. Invest., 93: 776 (1994)).

In addition, leukocyte infiltration upon intradermal injection of acompound into a suitable animal, such as rabbit, rat, or guinea pig, canbe monitored (see e.g., Van Damme J. et al., J. Exp. Med., 176: 59-65(1992); Zachariae, C. O. C. et al., J. Exp. Med. 171: 2177-2182 (1990);Jose, P. J. et al., J. Exp. Med. 179: 881-887 (1994)). In oneembodiment, skin biopsies are assessed histologically for infiltrationof leukocytes (e.g., eosinophils, granulocytes). In another embodiment,labeled cells (e.g., stably transfected cells expressing a CKR-3receptor, labeled with ¹¹¹In for example) capable of chemotaxis andextravasation are administered to the animal. Infiltration of cells inresponse to injection of a test sample (e.g., a compound to be tested ina suitable buffer or physiological carrier) is indicative of thepresence of a ligand or promoter, such as an agonist, in the sample.These assays can also be modified to identify inhibitors of chemotaxisand leukocyte extravasation. For example, an inhibitor can beadministered, either before, simultaneously with or after ligand oragonist is administered to the test animal. A decrease of the extent ofinfiltration in the presence of inhibitor as compared with the extent ofinfiltration in the absence of inhibitor is indicative of inhibition.

Diagnostic Applications

The present invention has a variety of diagnostic applications. Theseapplications include, but are not necessarily limited to theapplications discussed herein.

Mutation(s) in genes encoding a mammalian CKR-3 receptor protein cancause defects in at least one function of the encoded receptor, therebyreducing or enhancing receptor function. For instance, mutations whichproduce a variant of receptor or alter the level of expression, canreduce or enhance receptor function, reducing or enhancing, theinflammatory processes mediated by receptor.

For example, the methods of detecting or measuring receptor function canbe used to characterize the activity of receptors in cells (e.g.,leukocytes) of an individual or of receptors isolated from such cells.In these assays, reduced or enhanced receptor function can be assessed.

The nucleic acids of the present invention provide reagents (e.g.,probes, PCR primers) which can be used to screen for, characterizeand/or isolate a defective mammalian CKR-3 receptor gene, which encodesa receptor having reduced or enhanced activity. Standard methods ofscreening for a defective gene can be employed, for instance. Adefective gene and the activity of the encoded receptor can be isolatedand expressed in a suitable host cell for further assessment asdescribed herein for mammalian CKR-3 receptors. A number of humandiseases are associated with defects in the function of a G-proteincoupled receptor (Clapham, D. E., Cell, 75: 1237-1239 (1993); Lefkowitz,R. J., Nature, 365: 603-04 (1993)).

The antibodies of the present invention have application in proceduresin which receptor can be detected on the surface of cells. The receptorprovides a marker of the leukocyte cell types in which it is expressed,particularly in eosinophils. For example, antibodies raised against areceptor protein or peptide can be used to count cells expressingreceptor. Cell counts can be used in the diagnosis of a variety ofdiseases or conditions in which increased or decreased leukocyte celltypes (e.g., hypereosinophilia, for example in hypereosinophilicsyndrome; hypoeosinophilia) are observed. The presence of an increasedlevel of eosinophils in a sample obtained from an individual can beindicative of eosinophil infiltration due to an inflammatory disease orcondition, such as asthma, or an infection such as a parasiticinfections. Alternatively, or in addition, the antibodies can be used tosort cells which express receptor from among a mixture of cells.Suitable methods for counting and/or sorting cells can be used for thispurpose (e.g., flow cytometry, fluorescence activated cell sorting).

Furthermore, the antibodies can be used to detect or measure decreasedor increased expression of receptor in various diseases or conditions inwhich inflammatory processes of leukocytes are altered (e.g., increasedor decreased relative to a suitable control, such as the level ofexpression in a normal individual). For example, leukocytes (e.g.,eosinophils, lymphocytes such as T lymphocytes, monocytes, basophils)can be obtained from an individual and a suitable immunological assay(e.g., ELISA, FACS analysis) can be used to assess the level ofexpression. The level of expression of a mammalian CKR-3 receptor can beused in the diagnosis of a disease or condition in which increased ordecreased expression of a mammalian CKR-3 receptor is present.

Transgenic Animals

Transgenic animals, in which the genome of the animal host is alteredusing recombinant DNA techniques, can be constructed. In one embodiment,the alteration is not heritable (e.g., somatic cells, such as progenitorcells in bone marrow, are altered). In another embodiment, thealteration is heritable (the germ line is altered). Transgenic animalscan be constructed using standard techniques or other suitable methods(see e.g., Cooke. M. P. et al., Cell, 65: 281-291 (1991) regardingalteration of T lymphocytes; Hanahan, D., Science, 246: 1265-1275,(1989)).

In one aspect, an endogenous mammalian CKR-3 receptor gene can beinactivated or disabled, in whole or in part, in a suitable animal host(e.g., by gene disruption techniques) to produce a transgenic animal.Nucleic acids of the present invention can be used to assess successfulconstruction of a host containing an inactivated or disabled CKR-3 gene(e.g., by Southern hybridization). In addition, successful constructionof a host containing an inactivated or disabled CKR-3 gene can beassessed by suitable assays which monitor the function of the encodedreceptor.

In another embodiment, a nucleic acid encoding a mammalian CKR-3receptor protein or polypeptide is introduced into a suitable host toproduce a transgenic animal. In a preferred embodiment, endogenous CKR-3receptor genes present in the transgenic animals are inactivated (e.g.,simultaneously with introduction of the nucleic acid by homologousrecombination, which disrupts and replaces the endogenous gene). Forexample, a transgenic animal (e.g., a mouse, guinea pig, sheep) capableof expressing a nucleic acid encoding a mammalian CKR-3 receptor of adifferent mammalian species (e.g., a human) in leukocytes (such aseosinophils, lymphocytes (e.g., T lymphocytes) can be produced, andprovides a convenient animal model for assessing the function of theintroduced receptor. In addition, a compound can be administered to thetransgenic animal, and the effect of the compound on an inflammatoryprocess mediated by receptor can be monitored in a suitable assay ((seee.g., Weg, V. B. et al., J. Exp. Med., 177: 561 (1993); Abraham, W. M.et al., J. Clin. Invest., 93: 776 (1994)). In this manner, compoundswhich inhibit or promote receptor function can be identified or assessedfor in vivo effect.

Methods Of Therapy

Modulation of mammalian CKR-3 receptor function according to the presentinvention, through the inhibition or promotion of at least one functioncharacteristic of a mammalian CKR-3 receptor, provides an effective andselective way of inhibiting or promoting leukocyte-mediated inflammatoryaction. One or more ligands, inhibitors and/or promoters of CKR-3receptor function, such as those identified as described herein, can beused to modulate leukocyte function for therapeutic purposes.

As major eosinophil and lymphocyte chemokine receptors, mammalian CKR-3receptors provide a target for interfering with or promoting eosinophiland/or lymphocyte function in a mammal, such as a human. Consistently,co-localization of T cells and eosinophils is observed in certaininflammatory infiltrates. Thus, compounds which inhibit or promote CKR-3receptor function, such as ligands, inhibitors (e.g., 7B11) andpromoters identified according to the present method, are particularlyuseful for modulating eosinophil, basophil, and/or lymphocyte functionfor therapeutic purposes.

Thus, the present invention provides a method of inhibiting or promotingan inflammatory response in an individual in need of such therapy,comprising administering a compound which inhibits or promotes mammalianCKR-3 receptor function to an individual in need of such therapy. In oneembodiment, a compound which inhibits one or more functions of amammalian CKR-3 receptor (e.g., a human CKR-3 receptor) is administeredto inhibit (i.e., reduce or prevent) inflammation. As a result, one ormore inflammatory processes, such as leukocyte emigration, chemotaxis,exocytosis (e.g., of enzymes, histamine) or inflammatory mediatorrelease, is inhibited. For example, eosinophilic infiltration toinflammatory sites (e.g., in asthma) can be inhibited according to thepresent method.

In another embodiment, a compound which promotes one or more functionsof a mammalian CKR-3 receptor (e.g., a human CKR-3 receptor) isadministered to stimulate (induce or enhance) an inflammatory response,such as leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes,histamine) or inflammatory mediator release, resulting in the beneficialstimulation of inflammatory processes. For example, eosinophils can berecruited to combat parasitic infections.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals including, but not limited to, cows, sheep, goats,horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine,canine, feline, rodent or murine species can be treated. However, themethod can also be practiced in other species, such as avian species(e.g., chickens).

Diseases and conditions associated with inflammation and infection canbe treated using the method. In a preferred embodiment, the disease orcondition is one in which the actions of eosinophils and/or lymphocytesare to be inhibited or promoted, in order to modulate the inflammatoryresponse.

Diseases or conditions of humans or other species which can be treatedwith inhibitors of CKR-3 receptor function, include, but are not limitedto:

inflammatory or allergic diseases and conditions, including respiratoryallergic diseases such as asthma, allergic rhinitis, hypersensitivitylung diseases, hypersensitivity pneumonitis, eosinophilic pneumonias(e.g., Loeffler's syndrome, chronic eosinophilic pneumonia),interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, systemic lupuserythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren'ssyndrome, polymyositis or dermatomyositis); systemic anaphylaxis orhypersensitivity responses, drug allergies (e.g., to penicillin,cephalosporins), insect sting allergies; inflammatory bowel diseases,such as Crohn's disease and ulcerative colitis; spondyloarthropathies;scleroderma; psoriasis and inflammatory dermatoses such as dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria;vasculitis (e.g., necrotizing, cutaneous, and hypersensitivityvasculitis);

eosinphilic myositis, eosinophilic fasciitis;

autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis,multiple sclerosis, systemic lupus erythematosus, myasthenia gravis,juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis,Behcet's disease;

graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease;

cancers with leukocyte infiltration of the skin or organs;

other diseases or conditions in which undesirable inflammatory responsesare to be inhibited can be treated, including, but not limited to,reperfusion injury, atherosclerosis, certain hematologic malignancies,cytokine-induced toxicity (e.g., septic shock, endotoxic shock),polymyositis, dermatomyositis.

Diseases or conditions of humans or other species which can be treatedwith promoters of CKR-3 receptor function, include, but are not limitedto:

immunosuppression, such as that in individuals with immunodeficiencysyndromes such as AIDS, individuals undergoing radiation therapy,chemotherapy, therapy for autoimmune disease or other drug therapy(e.g., corticosteroid therapy), which causes immunosuppression;immunosuppression due congenital deficiency in receptor function orother causes;

infectious diseases, such as parasitic diseases, including, but notlimited to helminth infections, such as nematodes (round worms);(Trichuriasis, Enterobiasis, Ascariasis, Hookworm, Strongyloidiasis,Trichinosis, filariasis); trematodes (fluxes)(Schistosomiasis,Clonorchiasis), cestodes (tape worms)(Echinococcosis, Taeniasissaginata, Cysticercosis); visceral worms, visceral larva migrans (e.g.,Toxocara), eosinophilic gastroenteritis (e.g., Anisaki spp., Phocanemassp.), cutaneous larva migrans (Ancylostoma braziliense, Ancylostomacaninum).

Eosinophils as the Target Cell in Certain Inflammatory Reactions,Particularly Asthma

Eosinophils are produced in the bone marrow and circulate to thetissues, predominantly to mucosal tissues, such as the lungs,gastrointestinal tract, and genitourinary tract. Eosinophils typicallyconstitute 1-3% of leukocytes in the blood.

However, in people suffering from allergic diseases and helminthicparasitic infections, increased eosinophil accumulation occurs in thetissues or the blood. Eosinophils accumulation can be both beneficialand detrimental to the host.

For example, eosinophils possess numerous granules, containing cationicproteins. Degranulation of eosinophils, triggered, for example, by theengagement of IgG, IgA, or IgE receptors, or by stimulation byinflammatory mediators such as platelet-activating factor (PAF),leukotrienes, or chemokines, leads to release of the components in thegranule. Products from eosinophils also cause damage to host cells. Themost damaging are the cationic proteins, which are detectable inelevated concentrations in patients with asthma. Eosinophils alsogenerate a number of inflammatory mediators, including Leukotriene C4,and platelet-activating factor (PAF). These mediators contract airwaysmooth muscle, promote the secretion of mucus, alter vascularpermeability, and elicit further eosinophil and neutrophil infiltration.

Eosinophils are involved in the initiation and maintenance ofallergic/asthma diathesis. Thus, in a preferred embodiment, the methodcan be used to treat asthma or hypersensitivity (allergic) states,particularly those involving mucosal tissues, as well as in othereosinophil-associated diseases. In a particularly preferred embodiment,a compound which inhibits one or more function of a mammalian CKR-3receptor (e.g., a human CKR-3 receptor) is administered to an individualwith asthma.

Eosinophils are clearly important in the host defense against anddestruction of, large, nonphagocytable organisms, such as multicellularhelminthic parasites. Eosinophils are also important effector cells inimmune reactions against other pathogens that induce high levels of IgEantibodies. Accordingly, the method can be used to treat infectiousdiseases, such as parasitic diseases, to stimulate or promoteinflammatory defenses, or to suppress inflammatory responses which aredestructive to the host.

Eosinophils and Asthma Pathogenesis

Asthma is characterized by the obstruction of the airways or bronchi,and results from a bronchial hyperresponsiveness and rapid constrictionin response to a wide range of pharmacological mediators. Chronicinflammation of the bronchial mucosal lining is widely believed to playa fundamental role in the development of asthma.

Intense infiltration of the bronchial mucosa with eosinophils,macrophages and lymphocytes is observed in asthma and otherhypersensitivities. Often the selective migration of eosinophils toinflamed airways can be striking, and appears to result from theselective binding of eosinophils to endothelium and extraction from theblood. Eosinophils in particular are implicated as the causative agentsof bronchial mucosal injury. Studies of asthmatic patients suggest thatblood eosinophil counts correlate with the degree of bronchialhyperresponsiveness. In addition, bronchial biopsies and bronchoalveolarlavage fluid from asthmatics show a clear relationship between thedegree of eosinophilia and clinical severity. Thus, there is a strongconnection between the presence of eosinophils and adverse immunereactions, particularly in asthma.

A major chemokine receptor on eosinophils and lymphocytes, thatfunctions in selective leukocyte chemotaxis, extravasation andactivation in response to chemoattractant, provides an excellent targetfor interfering with eosinophil recruitment. For example, administrationof an inhibitor of at least one function of a mammalian (e.g., human)CKR-3 receptor, such as by inhibiting chemokine binding thereto, canprovide an effective and selective way of treating asthma. By reducingor preventing recruitment (extravasation, infiltration) of leukocytes,particularly eosinophils, to inflamed lung and airway tissues, and/orreducing leukocyte function in those tissues, the destructiveinflammatory processes of asthma can be inhibited, and the symptomsalleviated.

There is evidence that the blockage of eosinophil recruitment to thelung can alleviate the symptoms of asthma. Administration of amonoclonal antibody reactive with α4 integrin was reported to inhibitthe accumulation of eosinophils into the lung and airways, and blockedthe airway hyperresponsiveness to antigen challenge in sheep. In aprimate model of asthma, a monoclonal antibody to ICAM-1 is reported toattenuate airway eosinophilia and hyperresponsiveness. In addition, in aguinea pig model for passive cutaneous anaphylaxis, in vitropretreatment of eosinophils with the anti-α4 monoclonal was reported tosuppress eosinophil accumulation. (see Wegner, C. D. et al., Science,247: 456 (1990); Weg, V. B. et al., J. Exp. Med., 177: 561 (1993); andAbraham, W. M. et al., J. Clin. Invest., 93: 776 (1994) regarding thesemodels).

Modes of Administration

According to the method, one or more compounds can be administered tothe host by an appropriate route, either alone or in combination withanother drug. An effective amount of a compound (e.g., a receptorpeptide which inhibits ligand binding, an antibody or antibody fragment)is administered. An effective amount is an amount sufficient to achievethe desired therapeutic effect, under the conditions of administration,such as an amount sufficient for inhibition or promotion of a CKR-3receptor function, and thereby, inhibition or promotion, respectively,of an inflammatory response.

A variety of routes of administration are possible including, but notnecessarily limited to oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection),inhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops), routes of administration, depending on the disease orcondition to be treated. For respiratory allergic diseases such asasthma, inhalation is a preferred mode of administration.

Formulation of a compound to be administered will vary according to theroute of administration selected (e.g., solution, emulsion, capsule). Anappropriate composition comprising the compound to be administered canbe prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers (See, generally, Remington's Pharmaceutical Science, 16thEdition, Mack, Ed. 1980). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

EXEMPLIFICATION

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Example 1 Chemotactic Properties of Human Eosinophils

Chemotaxis Of Human Eosinophils

To identify antagonists of eosinophilic chemokine receptor(s), it isnecessary to identify the important chemokines for eosinophilchemotaxis, and determine the receptor(s) that these chemokines arebinding to. Chemotaxis experiments were performed in a sensitive andimproved chemotaxis assay, which employs an endothelial cell line grownon the polycarbonate membrane of the chemotaxis well.

Isolation Of Eosinophils

100 ml of heparinized blood was diluted 1:1 with PBS. 20 ml aliquotswere layered over 65%, 75% Percoll step gradients. The gradients werecentrifuged at 1500 rpm, 25 min at room temp. The eosinophil/neutrophillayers were transferred to a new tube and erythrocytes lysed by additionof 20 mls 0.2% NaCl for 1 min followed by the addition of 30 mls 1.8%NaCl. Cells were washed twice with a buffer consisting of PBS, 0.5% BSA,0.5 mM EDTA. Cells were resuspended at 5×10⁷ cells/50 μl in cold buffer(PBS, 0.5% BSA, 0.5 mM EDTA) and 50 μl CD16 microbeads were added to thecells. The mixture was incubated at 4° C. for 25 min followed by theaddition of 900 μl cold buffer. The miniMACS™ separation unit (MiltenyiBiotec, Inc., Auburn Calif. 95603) was used to deplete CD16 positivecells (neutrophils). Cells were loaded onto the column in 200 μlaliquots. Flow-through cells were collected and assessed histologically.The eosinophil prep was >99% pure.

Chemotaxis Assay

Chemokines were obtained from Peprotech, Inc. (Rocky Hill, N.J.).Chemotaxis experiments were performed using 3.0 micron BIOCOAT cellculture inserts (Collaborative Biomedical Products), in 24 well plates.Endothelial cells were grown to confluency on the inserts for two daysprior to chemotaxis experiments. The endothelial cells used were a cellline termed ECV 304 (European Collection of Animal Cell Cultures, PortonDown, Salisbury, U.K.), which expresses endothelial cell markers such asvon Willebrand factor, as well as ICAM-1 and VCAM-1. This endothelialcell line greatly facilitates these assays, since human umbilical veinendothelial cells can be variable in nature, can be used for onlyseveral passages, and grow much more slowly than ECV 304. The assay wasconducted at 37° C. for 1.5 hours, and migrated cells were counted usingan inverted microscope.

Results

The results, presented in FIG. 4, are representative of at least fiveexperiments. Growth of ECV 304 endothelial cells on the polycarbonatemembrane reduced the background migration almost completely. Eosinophilsapplied to transendothelial chemotaxis assays showed migration to anumber of chemokines, particularly RANTES, MCP-3, and to a lesser degreeMCP-1. MIP-1β, IL-8, MCP-2, and IP-10 had little effect on eosinophilchemotaxis. MIP 1α a was chemotactic for eosinophils in someexperiments, although generally was inactive. In these experiments, arange of chemokine concentrations was used, because of the variabilityin responsiveness of leukocytes to different chemokines, anduncertainties about the quality of chemokine preparations. A consistentfinding was the high level of eosinophil chemotaxis to RANTES and MCP-3.

Example 2 Identification of a Major Eosinophilic Chemokine Receptor

Primer Selection and Design

Five chemokine receptor genes were aligned and compared to generate aset of degenerate oligonucleotides for use in PCR (Polymerase ChainReaction) cloning of novel chemokine receptors from eosinophils. Theselection of these five receptor genes was based on either the type ofchemokine ligand with which they bind (Il-8 receptor A (IL8RA), Il-8receptor B (IL8RB), MIP-1α receptor (MIP1αR)) or orphan receptors withsignificant sequence similarity to these receptors whose expression isreported to be restricted to lymphoid cells or tissue (Epstein BarrInducible receptor-1 (EBI1R) and Burkitt's Lymphoma Receptor-1 (BLR1)).Receptor sequences were aligned by hand based on a number of publishedalignments (IL-8RA, Holmes et al., Science, 253: 1278-1280 (1991);IL-8RB, Murphy, P. A. et al., Science, 253: 1280-1283 (1991);MIP1α/RANTES, Neote, K. et al., Cell, 72: 415-425 (1991); EBI1R,Birkenbach, M. et al., J. Virol., 67: 2209-2220 (1993); and BLR1(Dobner, T. et al., Eur. J Immunol., 22: 2795-2799 (1992)).

Sequences within transmembrane (TM) regions 2, 6 and 7 as well as aregion just C-terminal to TM3 were selected as targets for degenerateoligonucleotide design based on the high degree of sequence similarity.The nucleotide sequences of the degenerate oligonucleotide primers areillustrated in the Table below. TABLE Primer Set 2 SEQ ID NO: TM2a 7Primer 2a-1 (forward) 5′- TAC CTG CTS AAC CTG CCC ITG GCI G 8 Nestedprimer 2a-2 (forward)              5′- AC CTG GCC ITG GCI GAC CTM CTC TTTM3 9 Primer 3F (forward) 5′- GAC CGY TAC CTG GCC ATI GTC CAY GCC 10Primer 3R (reverse)    CTG GCR ATG GAC CGG TAI CAG GTR CGG-5′ TM6b 11Primer 6b-1 (reverse)         GAR AMR ACC IRI GGG ATG TTR IAC CAI-5′ 12Nested primer 6b-2 (reverse) AAG RAI GAR GAR AMR ACC IRI GGG ATG T-5′TM7 13 Primer 7-1 (reverse)         ACG SAG TTG GGI IAS IAG ATG CGGAAG-5′ 14 Nested primer 7-2 (reverse) GTG WCG ACG SAG TTG GGI IAS IAGA-5′Nucleotide Abbreviations:K = G/TM = A/CR = A/GS = C/GW = A/TY = C/TEosinophil Isolation and Purification

100 ml of heparinated blood was diluted 1:1 with PBS. 20 ml aliquotswere layered over 65%, 75% Percoll step gradients. The gradients werecentrifuged at 1500 rpm, 25 min at room temperature. Theeosinophil/neutrophil layers were transferred to a new tube anderythrocytes lysed by addition of 20 mls 0.2% NaCl for 1 minute followedby the addition of 30 mls 1.8% NaCl. Cells were washed twice with asolution of phosphate buffered saline (PBS), 0.5% Bovine Serum Albumin(BSA), 0.5 mM ethylenediaminetetraacetic acid (EDTA). Cells wereresuspended at 5×10⁷ cells/50 μl in cold buffer (PBS, BSA, EDTAsolution), and 50 μl CD16 microbeads were added to the cells. Themixture was incubated at 4° C. for 25 min followed by the addition of900 μl cold buffer. The miniMACS™ separation unit (Miltenyi Biotec,Inc., Auburn, Calif. 95603) was used to deplete CD16 positive cells(neutrophils). Cells were loaded onto the column in 200 μl aliquots.Flow-through cells were collected and assessed histologically. By thiscriteria, the eosinophil prep was >99% pure.

mRNA Isolation and PCR

mRNA for RT-PCR (Reverse transcription-polymerase chain reaction) wasextracted directly from purified cells using the Micro-FastTrack™ mRNAisolation kit purchased from Invitrogen. Quality of the mRNA wasevaluated by PCR amplification of β-actin and/or GAPDH(glyceraldehyde-3-phosphate dehydrogenase) mRNA prior to use with 7TMSdegenerate primers.

20-50 ng of mRNA was reverse transcribed using a GeneAmp® RNA PCR kit(Perkin-Elmer) with oligo dT and/or random hexamers as primers in a 20μl final volume as specified by the manufacturer. 2-5 μl of this cDNA(reverse transcribed eosinophil message) was mixed with 200 μM dNTPs and50-100 pmol of degenerate primers in a 50 μl volume. Magnesiumconcentration and pH were optimized for each primer pair. The magnesiumconcentration ranged from 1.0 to 3.0 mM and pH ranged from 8.5 to 10.0.Although various cycle parameters were also evaluated, the conditionsgenerally used were similar to the following:

3 cycles: 94° C., 30 sec; 37° C., 30 sec; 2 min ramp to 72° C., 1 min,followed by 30 cycles: 94° C., 45 sec; 48° C., 1 min; 72° C., 1 min.(ramp=gradual increase).

With regard to the 201 bp fragment isolated (see below), primer pairs2a-1 and 7-1, or primer pairs 2a-1 and 3R, were used in a PCR reaction(as described above) in 60 mM Tris-HCl, pH 9.5 and 1.5 mM MgCl₂. One μlof product from each reaction was used in a separate (second) round ofPCR with “nested” primers 2a 2 and 3R. (“Nested” primers are primerswhich hybridize to sequences within the outside primers.) Reactionconditions for the nested PCR were exactly as described for the firstPCR.

PCR products were assessed and separated by agarose gel electrophoresis,and appropriately sized fragments were purified and subcloned using thepCR-Script™ SK+ cloning kit (Stratagene). (Appropriate fragment sizesare as follows: for PCR with primer pairs from regions 2a and 7 (seeTable above), ˜700 bp; for PCR with primers from region 2a and primer3R, ˜200 bp; for PCR with primer 3F and primers from region 6b, ˜400 bp,and for PCR with primer 3F and region 7 primers, ˜550 bp.) Expectedfragment sizes were predicted based upon the hypothesis that a relatedreceptor protein would share some structural similarity.

Rapid Screening Assay

In order to screen a large number of clones quickly for novel members ofthe 7TMS family, the inserts of bacterial colonies obtained as describedabove (i.e., transformants of plasmids comprising appropriately sizedfragments subcloned into pCR-Script SK+), were screened by PCR using T3and KS primers complementary to the sequence flanking the polylinker ofpCR-Script™. In particular, a portion of a bacterial colony from anovernight transformation was mixed directly with 40 μl of a PCR mixturecontaining 200 μM dNTPs, 20 mM Tris, pH 8.5, 50 mM KCl, 2.5 mM MgCl₂, 50pmol each primers and 0.25 units Taq polymerase. Cycle conditions were25 cycles: 94° C., 20 sec; 55° C., 20 sec; 72° C., 30 sec. Inserts ofthe correct size were identified by evaluating 20 μl of PCR product on1.5% agarose gels. The remaining 20 μl of the reaction was digested withAlu I, Hha I, and Rsa I (triple digestion) and resolved on a 12%polyacrylamide gel to screen for different digestion patterns. Clones ofdifferent patterns were then selected for sequence analysis.

Results

Sequence analysis of PCR fragment, generated from degenerate oligos,identified a 201 bp partial cDNA clone in pCR-Script. (The degenerateoligos were 2a-1, 2a-2, 3F, 3R and 7-1). This partial clone, designatedEos L2 (also referred to as L2 and EL2), was found to have 78.3% aminoacid similarity (81.1% nucleic acid similarity) to the MIP1α/RANTESreceptor and 60.8% amino acid similarity (61.6% nucleic acid similarity)to the MCP-1 receptor. A search of the most current sequence data basesrevealed this partial clone to be unique.

Southern and Northern Analysis

The PCR fragment was labeled and used to probe both Southern andNorthern blots. To prepare the PCR probe, the 201 bp fragment wasreleased from the pCR-Script vector with restriction enzymes EcoRI andNot I. This digested resulted in a fragment of 240 bp comprised of the201 bp fragment plus 39 base pairs of polylinker from the vector. Thefragment was separated from vector by electrophoresis through agarosegel, and purified by (Magic Mini Prep, Promega Corp. Madison, Wis.)exactly as recommended by the manufacturer. Approximately 200 ng ofmaterial was labeled with the Random Primed DNA Labeling Kit purchasedfrom Boehringer Mannheim following the manufacturer's recommendedlabeling protocol.

For Southern blots, genomic DNA (purchased from Clontech Laboratories,Inc., Palo Alto, Calif.) was digested with restriction enzyme overnightand separated by electrophoresis on a 0.7% agarose gel followed bycapillary transfer to Hybond-N nylon membrane (Amersham). Hybridizationwas in 6×SSC (1×SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)containing 5× Denhardt's solution (1× Denhardt's solution is 0.02%bovine serum albumin, 0.02% ficoll, 0.02% polyvinylpyrolidone), 10% w/vdextran sulfate, 2% SDS, and sheared salmon sperm DNA (100 μg/ml)overnight at 65° C. The membrane was rinsed twice in 2×SSC, 0.5% SDS at65° C. followed by two washes (15 min each) in 0.2×SSC, 0.5% SDS at 65°C.

The Southern hybridization revealed a single strongly hybridizingfragment and a single weakly hybridizing fragment with each enzyme used.The weakly hybridizing fragment is likely to be the MIP1α1/RANTESreceptor.

Multiple Tissue Northern Blots were purchased from ClontechLaboratories, Inc., Palo Alto, Calif.). ExpressHyb™ Solution was alsopurchased from Clontech Laboratories, Inc. The Multiple Tissue NorthernBlots were carried out as recommended by the manufacturer. The probe wasas described above for Southern blots. The results of the Northernhybridization showed high levels of a˜1.6 kb message in spleen,peripheral blood leukocytes and thymus. Additional Northern analyses arepresented in Example 5.

Genomic Library Screening

A human genomic phage library constructed in the EMBL3 SP6/T7 vector,purchased from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), wasscreened with the 201 bp PCR fragment to obtain a full-length clone.Approximately 25,000 plaque forming units were mixed with 600 μl of anovernight bacterial culture of E. coli strain K802 provided with thelibrary in NZCYM top agarose and plated on 150 mm petri dishescontaining NZCYM agar (NZYCM broth, Agar and Agarose were purchased fromGibco/BRL). After incubation at 37° C. for 7 hours, the plates wereoverlaid with BA-85 nitrocellulose membranes (Schleicher and Schuell,Keene, N.H.) for 5 minutes to allow transfer of phage to membrane. Themembranes were then soaked for 5 minutes in Denturing Solution (1.5 Msodium chloride, 0.5 N sodium hydroxide) followed by neutralization in1.5 M sodium chloride, 0.5 M Tris, pH 8.0. The filters were allowed toair dry for 15 minutes and then baked for two hours at 80° C. undervacuum. The filters were then hybridized as described above for theSouthern Blot. The 201 bp PCR fragment contained the nucleotides betweenoligonucleotide primers 2a-2 (TM2) and 3R (TM3).

One genomic phage clone, designated Eos L2.8, contained an insert whichcomprises the 1.8 kb Hind III fragment seen on Southern blots (completeinsert size was not determined, but is ˜17 kb).

Phage clone Eos L2.8 was digested with Hind III restriction enzyme andelectrophoresed on an agarose gel. A Hind III fragment of approximately1.8 kb was cut out, electroeluted from agarose, phenol/chloroformextracted and precipitated with ethanol. The 1.8 kb fragment wasresuspended in water and ligated into the Hind III site of thepBluescript II KS+ vector (Stratagene) followed by transformation intoDH5α competent cells purchased from Gibco/BRL.

Both strands of this Hind III fragment were sequenced, and the fragmentwas found to contain the entire amino acid coding region for the Eos L2receptor (a human CKR-3 receptor). Comparison of this sequence and thecDNA clone described below indicates that the clone is a full-lengthclone. The open reading frame of 1065 nucleotides encodes a protein of355 amino acids (SEQ ID NO:2) with a predicted molecular mass of 41 Kd.

Comparison of the sequence of the full-length Eos L2 receptor withMIP1α/RANTES and MCP-1 receptors revealed a 73.4% and 60.5% amino acidsimilarity, respectively. For this comparison, sequences were aligned byhand and the number of similar amino acids, divided by the total numberof amino acids was multiplied by 100.)

The sequences were also aligned by the Clustal method using MegAlign™(DNASTAR, Inc.). Comparison with other chemokine receptor sequencesrevealed a 62%, 47%, and 41% amino acid sequence similarity to CKR-1,CKR-2B, and CKR 4, respectively. In contrast, the amino acid sequencesimilarity to IL-8 receptors A and B was only 27% for both receptors.The sequence similarity of this receptor to MIP1α/RANTES and MCP-1receptors, both C—C chemokine receptors, is consistent with the resultsreported herein which indicate that Eos L2 is a C—C chemokine receptor.

Example 3 Expression of Eos L2 in Transfected Cell Lines

FLAG-Tagged Eos L2 (CKR-3) Receptor Construct

An Eos L2 receptor fusion protein was constructed as follows:

1. A FLAG-PAF receptor construct in pCDM8 (constructed as reported inKunz, D. et al., J. Biol. Chem., 267: 9101-9106 (1992)) was doubledigested with Hind III and EcoRI to release a 48 bp fragment containingnucleotides which encode the FLAG peptide. The nucleotide sequence isAAGCTTCCA GCA GCC ATG GAC TAC AAG GAC GAC GAT GAC AAA GAATTC (SEQ IDNO:15). The amino acid sequence is MDYKDDDDKEF (SEQ ID NO:16). The 48 bpHind III/EcoRI fragment containing the FLAG nucleotides subcloned intothe HindIII/EcoRI sites of the pcDNA3 vector (Invitrogen, San Diego,Calif.) giving rise to pcDNA3/FLAG.

2. The pBluescript II KS+ vector containing the 1.8 kb Eos L2 Hind IIIfragment was digested with BamHI and Xho I to release a 1.261 kbfragment. This BamHI-XhoI fragment contains nucleotides encoding Eos L2amino acids 91 through the stop codon plus the same 3′ untranslatedregion and 21 bp of pBluescript II KS+ vector.

3. Two PCR primers were generated to amplify the 5′ end of the Eos L2gene, but removing the first Met and engineering in an EcoRI site whichwill be compatible with the EcoRI site described above in step 1. The 5′primer (SEQ ID NO:17) was:         EcoRI 5′-TTAA GAATTC ACA ACC TCA CTAGAT AC

This primer contains an EcoRI site and the first 17 nucleotides of theEosL2 gene except for the Met codon.

The 3′ primer (SEQ ID NO:18) was:            BamHI 5′-CATAGT GGATCCAGAATG

This primer primes in the Eos L2 gene just 3′ to the BamHI site.Amplification with these two primers using the pBluescript II KS+ vectorcontaining the 1.8 kb Eos L2 fragment as template will amplify a 280 bpfragment containing the 5′ end of the Eos L2 which can be digested withEcoRI and BamHI to give a fragment for ligation as described below.

Conditions for amplification were: 100 ng of pBluescript II KS+containing the 1.8 kb EosL2 fragment was combined with 200 μM dNTPs and50 pmol of primers in a 50 μl reaction volume. The final magnesiumconcentration was 2.5 μM and the pH was 8.0. The fragment was amplifiedwith 25 cycles of 94° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec. Theamplified product was separated on agarose gel and purified byelectroelution as described above. The fragment was digested with EcoRIand BamHI purified again on agarose gel.

4. For construction of the Flag-tagged EosL2 gene, the pcDNA3 vectorcontaining the FLAG fragment (described in step 1) was digested withEcoRI and Xho I. The vector fragment (an EcoRI-XhoI fragment comprisingthe FLAG coding sequence) was separated from the polylinker fragment byelectrophoresis, and the vector fragment was purified as described forother electroeluted fragments. The vector fragment was combined with theEcoRI-BamHI fragment generated by PCR in step three. These two fragmentswere combined with the 1.261 kb BamHI-XhoI fragment from step two. Allthree fragments were triple ligated together to yield the FLAG-taggedEos L2 receptor in pcDNA3. Ligated DNA was transformed into DH5α.

Transient Transfectants

293 cells (ATCC Accession No. CRL 1573) were grown in Minimal EssentialMedium (MEM) Alpha Medium obtained from Gibco/BRL and supplemented with10% fetal Calf Serum, Glutamine, and Penicillin/Streptomycin (all fromGibco/BRL). For each transient transfection, 2×10⁶ 293 cells were plated1 day before transfection in a 35-mm tissue culture dish. On the day oftransfection, the cells (which grow attached to the dish) were washed 1×with Phosphate Buffered Saline (PBS, Gibco/BRL) and a mixture of DNA andlipofectAMINE™ Reagent (Gibco/BRL) were applied to the cells.

The DNA/lipofectAMINE™ reagent mixture was made by incubating 2 μg ofFlag-tagged Eos L2 receptor expression vector in a final volume of 100μl OptiMEM™ (Gibco/BRL) with 12 μl of LipofectAMINE™ reagent in a 100 μlvolume for 45 minutes at room temp. The final mixture volume is 200 μl.After the 45 minute incubation, 800 μl of OptiMEM™ is added to the 200μl of DNA/lipofectAMINE™ reagent and the 1 ml of solution is layeredover the cells as described above. The cells were then incubated at 37°C. for 5 hours at which time 1 ml of MEM Alpha Medium supplemented asdescribed above is added. The cells are incubated for an additional 12hours at which time all medium is removed and the cells washed 2× withPBS and 2 mls of MEM Alpha medium supplemented as described above isadded. The transfected cells are then incubated for an additional 72hours. The cells are harvested by gently pipetting them after incubationin PBS 10 mM EDTA.

Cell surface expression of a FLAG-tagged Eos L2 receptor wasdemonstrated in the transiently transfected 293 cells. Approximately2.6% of the cells express the receptor on the surface as determined byimmunofluorescent staining and FACS analysis. Levels of expression insome cells were found to be as much as 2 logs greater than backgroundindicating that high levels of expression can be achieved in this cellline. As the Eos L2 gene is carried by the pcDNA3 expression vector(Invitrogen Corp., San Diego, Calif.), which contains the neomycinresistance gene, stable 293 transfectants can be selected usinggeneticin (G418) selection.

Stable Cell Lines

Over 500 stable lines of mouse L1-2 pre-B cells have been generated withthe FLAG-tagged receptor. L1-2 pre-B cells were obtained from (Dr.Eugene Butcher, Stanford University, Stanford, Calif.), and weremaintained in RPMI-1640 (Gibco/BRL), supplemented with 10% bovine serumalbumin, and Pen/Strep, sodium pyrvate and β-mercaptoethanol. Cells fromover 200 clones were screened for surface expression by staining with M2anti-FLAG monoclonal antibody (International Biotechnologies, Inc., NewHaven, Conn.), followed by anti-mouse Ig-FITC (Jackson ImmunoResearchLaboratories, Inc.), and analyzed by fluorescence activated cell sorting(FACS). Immunofluorescent staining and FACS analysis was performed asdescribed in Current Protocols in Immunology, Vol. 1, Coligan, J. etal., Eds., (John Wiley & Sons, Inc.; New York, N.Y.). Results of theFACS analysis for several cell lines revealed a number of clones whichexpress high levels of the Eos L2 flagged receptor (FIG. 5).Untransfected cells (not shown) were negative for staining. Stable celllines with high level expression can be used as immunogens for theproduction of antibodies reactive with the Eos L2 receptor. In addition,these cell lines are useful for studying chemotaxis and ligand binding.

Baculovirus Expression

For construction of a baculovirus expression vector, the Flag-tagged EosL2 receptor in pcDNA 3 was digested with HindIII to remove theFlag-tagged gene. The HindIII fragment containing the gene was bluntended by filling in the overhangs with Klenow fragment and dNTP's. Theblunt ended fragment was subcloned into the Sma I site of pVL1393(Invitrogen). 2.0 μg of the pVL1393 vector containing the Eos L2 genewas mixed with 0.5 μg of AcMNPV viral DNA (Invitrogen) andco-transfected into Sf9 insect cells (Invitrogen) with Insectin™(Invitrogen) according to the manufacturer's instructions. The SF-900media (serum free) was replaced with 5 ml of SF-9 culture medium(Grace's Supplemented Insect Media (Gibco/BRL) containing 10% fetal calfserum) on the following day, and the cells were allowed to grow for fivedays. Recombinant virus was plaque purified as described in D. R.O'Reilly, L. K. Miller, and V. A. Luckow (1994) Baculovirus expressionvectors: A Laboratory Manual, Oxford University Press, pp. 149-158.

Expression of the Eos L2 receptor was obtained on Sf9 cells by infectingSf9 cells with the plaque purified recombinant virus described above.The Sf9 cells (2×10⁶ cells/ml) were infected at a multiplicity ofinfection of 10:1. The infection proceeded for 72 hours at which timethe cells were stained with the M2 anti-FLAG antibody.

Successful expression of this receptor was also achieved with abaculovirus expression system in Sf9 cells. Good levels of expressionhave been achieved based on staining with anti-FLAG antibody (seeExample 5). Ligand binding was also achieved with the same cells Sf9transfectants shown by FACS to be expressing receptor. While definitivecell surface expression was shown by propidium iodide exclusion,expression on these cells appeared to be low, as compared with anegative control (i.e., Sf9 cells transfected with expression vectorlacking the Eos L2 gene insert). Length of infection can be decreased,and MOI can be further optimized, for higher cell surface expression.

Example 4 Ligand Binding Studies

Ligand Binding Procedure

Cells transfected with Eos L2 receptors or normal human eosinophilspurified (see above) were washed in Hanks Balanced Saline Solution(HBSS), then resuspended in binding buffer: 50 mM HEPES, 1 mM CaCl₂, 5mM MgCl₂, 0.5% Bovine Serum Albumin (BSA), pH 7.3. In microfuge tubes,5×10⁵ cells were incubated with 0.1 nM radiolabeled chemokine (purchasedfrom New England Nuclear, Massachusetts) in 200 μl aliquots at roomtemperature for 60 minutes. The cells were either incubated withradiolabeled chemokine alone, or together with unlabeled chemokines(from PeproTech) as competitors, which were used at the indicatedconcentrations. At the end of incubation, cells were washed 3 times inthe binding buffer, each wash consisting of centrifugation in amicrofuge at 7,000×g for 2 minutes. After the wash, the pellets weretransferred into LP3 tubes and the radioactivity of the cells, whichrepresented the amount of binding was measured in a gamma counter. Allsamples were in duplicates and all the experiments were repeated atleast 3 times. Scatchard Plot was calculated from the binding data byMicroSoft Excell and CricketGraph on a Macintosh computer.

Binding To Human Eosinophils

Based on the findings from chemotaxis assays (see Example 1), the ligandbinding studies focused on RANTES, MIP-1α and MCP 3. The ligand bindingstudies were carried out using radiolabeled chemokines and various‘cold’ chemokines as competitors. Purified normal human eosinophils wereincubated with either 0.1 nM ¹²⁵I labeled MIP-1α or RANTES in thepresence or absence of various cold chemokines (250 nM MIP-1α, RANTES,IL-8, MCP-1 or MCP-3). After extensively washing the cells, the bindingwas measured by a gamma counter.

FIG. 6 is a histogram illustrating the binding of human eosinophils toRANTES and MIP-1α. These results suggest that eosinophils bind onlyweakly to MIP-1α, and that this binding can be inhibited by MIP-1αitself and by other β-family chemokines, e.g., MCP-1, MCP-3 and RANTES(FIG. 6). In contrast, eosinophils bound RANTES more abundantly (FIG.6). Binding by RANTES could not be inhibited efficiently by excessamount of ‘cold’ MIP-1α (FIG. 7), suggesting that on eosinophils, therecould be distinguished receptors for MIP-1α and RANTES.

Scatchard plot analysis revealed that there are 1.8×10³ MIP-1α bindingsites with an affinity of 91 pM. The analysis also revealed a loweraffinity (883 pM) receptor for RANTES, having more binding sites(3.6×10⁴/cell). Under the conditions used, there was no significantMCP-1 binding to eosinophils (not shown), and MCP-1 did not inhibitRANTES binding except at very high concentrations (2500-fold excess,FIG. 7).

Eos L2 Receptor Transfectants

Following the cloning and expression of the Eos L2 receptor, transfectedcells were used to test binding to a number of chemokines. The firstattempts using 293 transfectants were unsuccessful, as the addition ofcold chemokines interfered with binding, a phenomenon observed by otherinvestigators. In contrast, using baculovirus infected SF9 cells, goodRANTES binding could be detected (FIG. 8). The assay conditions for SF9cells were different from that of mammalian cells. Binding of 0.1 nM¹²⁵I labeled RANTES took place in 50 mM HEPES, pH 7.3, 5 mM MgCl₂ and 1mM CaCl₂, supplemented with 0.5% BSA. After 60 minutes at roomtemperature, the cells were washed three times in the binding buffercontaining 0.5 M NaCl, and the radioactivity in the cell pellets wascounted using a gamma counter.

In these ligand binding assays, the most effective heterologouscompetitor of MIP-1α or RANTES binding was MCP-3. In fact, MCP-3 alsoeffectively inhibited MCP-1 binding to activated T cells. Thus, MCP-3appears to bind to CKR-1, CKR-2 and CKR-3 (CKR-1, Gao, J. L., et al., J.Exp. Med., 177: 1421-1427 (1993) and Neote, K., et al., Cell, 72:415-425 (1993); CKR-2, Charo, I. F., et al., Proc. Natl. Acad. Sci. USA,91: 2752-2756 (1994) and Myers, S. J., et al., J. Biol. Chem., 270:5786-5792 (1995)).

Radiolabeled MCP-3 (Peprotech, Inc. Rocky Hill, N.J.) was also used forbinding studies. MCP-3 binding was carried out as described above withthe following modifications. Cells were incubated with 0.1 nM¹²⁵I-labeled MCP-3. The binding buffer used was HBSS plus 0.5% BSA and0.1% sodium azide. Binding took place at 37° C. for 30 min. The unboundisotope was separated by spinning cells through 800 μl of 20% sucrose,at 12,000×g for 2 min. The tubes were then snap-frozen in dry ice, thetips cut off with a pair of pliers and counted.

Example 5 Expression of the Eosinophilic Chemokine Receptor

To confirm that the Eos L2 receptor is the functional receptor oneosinophils, the expression of the receptor was assessed by (a) Northernblot analyses, and (b) flow cytometry using monoclonal antibodiesanti-peptide antibodies reactive with the receptor.

Purification of Human Eosinophils, Neutrophils, and PBMC

Eosinophils were isolated from heparinized blood of individuals withhigh levels of circulating blood eosinophils (5-17%) by combined densitygradient centrifugation and negative selection with anti-CD16 magneticbeads (Hansel, T. T. et al., J. Immunol. Meth., 122: 97 (1989)).Briefly, the granulocyte fraction from the Percoll centrifugation wasincubeated with CD16 microbeads (Miltenyi Biotec, Inc., Sunnyvale,Calif.) for 30 minutes. Cells were then passed through a MACS column(Miltenyi Biotec, Inc.), and eosinophils were collected in theflow-through. Eosinophils were shown histologically to be >99% pure asdetermined by analysis of Diff-Quick-stained cytocentrifugationpreparations by light microscopy.

Human neutrophils were isolated from heparinized venous blood by Percolldensity gradient centrifugation (δ=1.088) at room temperature (Coliganet al., Eds., 1992, Current Protocols in Immunology, (John Wiley & Sons:New York, N.Y.)). RBCs were removed by hypotonic lysis. PBMCs were alsoisolated as described (Coligan et al., Eds., 1992, Current Protocols inImmunology, (John Wiley & Sons: New York, N.Y.)). Monocytes werepurified by CD14 positive selection with magnetic beads and T cells werepurified by passage of lymphocytes over nylon wool. To generate CD3blasts, 2×10⁶ PBMCs/ml in RPMI-1640 plus 10% FCS were added to tissueculture plates first coated with the anti-CD3 antibody TR77. After 4-6days blasts were removed to fresh media and supplemented with IL-2(Genzyme) at 50 units/ml.

Northern Analyses: CKR-3 is Expressed Selectively in Eosinophils

Although eotaxin is a selective chemoattractant for eosinophils, theCKR-3 receptor also binds RANTES and MCP-3, which are known to attractmonocytes and T cells. Message expression of the receptor was examinedin various leukocyte populations.

The results of initial Northern hybridization (see Example 2) showedexpression of a ˜1.6 kb message in spleen, peripheral blood leukocytes,and thymus, and a number of leukocyte subpopulations, such aseosinophils and T cells, as well as in the HL-60 cell line. Messagelevels increased dramatically in the HL-60 cell line upon butyric acidinduction down the eosinophilic pathway.

This message is likely to be that of Eos L2, since the message for theMIP1α/RANTES receptor which cross-hybridizes on Southern blots is weakand is reported to be approximately 3.0 kb. When the original 201 bp PCRfragment is used as a probe in Southern blots, a strongly hybridizing1.8 kb HindIII fragment is seen. This is the fragment that was clonedand discussed here. In addition to this fragment, a very weaklyhybridizing fragment at about 10 kb is observed. This 10 kb fragmentcorresponds to the reported HindIII fragment size of the MIP1α/RANTESreceptor. This MIP1α/RANTES receptor produces a message of approximately3 kb which is not observed on Northerns. Therefore, the ˜1.6 kb messageseen on Northerns probably derives from Eos L2 gene. By far the mostabundant expression of Eos L2 was observed in a preparation of purifiedeosinophils from a patient with hyper-eosinophilic syndrome (see Example8).

Because of the high sequence similarity of CKR-3 to other CC chemokinereceptors and the fact that the full-length clone hybridizes to multiplesequences in Southern blots, additional Northern analyses used a 250 bpfragment from the 3′ untranslated region of the genomic clone which doesnot cross-hybridize with other sequences in Southern blots. Forhybridization, a 3′-untranslated region probe specific for CKR-3 wasused encompassing nucleotides 1203-1453 (FIG. 1C).

A Northern blot panel was prepared using RNA from different leukocytepopulations, including monocytes, neutrophils, lymphocytes, T cells, Tcell blasts produced by activation with CD3 MAb, and eosinophils. RNAwas isolated using TriZOL™ reagent (Gibco/BRL) following themanufacturer's recommended protocol. 15 μg of total RNA isolated fromeach highly purified leukocyte population was separated on 1.2%formaldehyde agarose gels and transferred to Nytran-Plus™ nylon membrane(Schleicher and Schuell) and cross-linked using a Stratalinker®.Hybridization with radiolabeled 3′-untranslated region probe was withExpressHyb™ Solution (Clontech) using the manufacterer's suggestedprotocol. Northern blots were exposed to X-OMAT AR film for 3-5 dayswith intensifying screen. CKR-3 specific probe was removed by boiling in0.5% SDS and the blot re-probed with β-actin to control for variation inloading.

The only cell population which gave a detectable signal was eosinophils,where a message 1.8 kb in size was found. These results are consistentwith the pattern of surface expression detected immunologically in FIGS.13A-13D. Although message was not detected in resting or activated Tcells in this experiment, it is possible that a subset of T cells mayexpress the receptor.

Monoclonal Antibodies (MAbs) Reactive with the Eosinophilic ChemokineReceptor

MAbs reactive with the Eos L2 receptor were generated by immunizing micewith a synthetic peptide corresponding to the N-terminal 35 amino acids.The N-terminal 35 amino acids of Eos L2, deduced from the nucleotidesequence (see FIGS. 1A-1D; see also, SEQ ID NO:2), were synthesized andcoupled to the carrier protein PPD (Purified Protein Derivative ofMycobacterium tuberculosis; Severn Biotech Ltd., Cambridge, U.K.).

Female Balb/C mice were immunized with 50 μg of this peptidepeptide-carrier conjugate in PBS 4 times at 2 week intervals. Mice wereinjected intra-peritoneally with the peptide conjugate, using Freund'scomplete (first injection) and incomplete adjuvant (subsequentinjections). The final immunization was injected intravenously withoutadjuvant. Polyclonal antiserum was also collected from mice immunizedwith synthetic peptide.

Two successful fusions were performed which generated over 15,000hybridomas. Four days after the final injection, the spleen was removedand a single cell suspension prepared in serum free DMEM media. Thesecells were fused with the hybridoma fusion partner SP2/0, according toGalfre, G. et al. (Galfre, G. et al., Nature, 266: 550-552 (1977)). 20ml of spleen cells and 20 ml of SP2/0 were combined, spun at 800 g for 5min and the media removed. A solution of 50% Polyethylene glycol 1500(Boehringer Mannheim, Indianapolis, Ind.) prewarmed to 37° C. was addedto the cell pellet over 2 min, followed by 10 ml of DMEM media over 3min. The cell suspension was spun at 400 g for 3 min and the supernatantremoved. The pellet was resuspended gently in DMEM media containing 20%fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/mlstreptomycin sulfate, and HAT selection media (Boehringer Mannheim,Indianapolis, Ind.). Cells were plated into 96 well flat bottommicrotiter plates at 200 μl/well.

10-14 days later, supernatants from the wells were screened forreactivity against the peptide using an enzyme-labeled anti-mouseantibody (Horseradish peroxidase-labeled anti-mouse IgG (Jackson) in anELISA assay. Approximately 200 mAbs were selected that showed strongreactivity against the synthetic peptide. Hybridomas of interest weresubcloned using limiting dilution.

To determine which antibodies could recognize the native, surfaceexpressed molecule, the MAbs were screened against Sf9 insect cellsinfected with AcMNPV virus carrying human Eos L2 genomic DNA. Theseinsect cells expressed Eos L2 (CKR-3) receptor on the cell surface, asjudged by strong anti-FLAG staining of approximately 10% of cells.Staining was performed using M2 anti-FLAG antibody, followed byanti-mouse Ig-FITC (Jackson ImmunoResearch Laboratories, Inc.), andanalyzed by flourescence activated cell sorting, using FACScan analysisto quantitate expression. (Current Protocols in Immunology, Vol. 1,Coligan, J. et al., Eds., (John Wiley & Sons, Inc.; New York, N.Y.).

Approximately 33% of the anti-peptide hybridomas reacted with the Eos L2transfected insect cells, with a staining pattern identical to that ofthe FLAG antibody, as determined by FACS analysis using anti-mouseIg-FITC (Jackson ImmunoResearch Laboratories, Inc.) as second antibody.Untransfected insect cells stained with anti-FLAG were completelynegative. Anti-peptide antibody also tested against untransfected cells,which were negative for staining.

MAbs that were found to stain the transfected insect cells were examinedusing FACS analysis for their reactivity with human eosinophils,peripheral blood lymphocytes, monocytes, neutrophils, and activated Tcells (activated T cells; lymphocytes were treated with an anti-CD3antibody to activate T cells). Cells were stained with mAb LS26-5H12 andthen FITC-anti-mouse Ig (Jackson ImmunoResearch Laboratories, Inc.). Fcreceptor binding was controlled for by using an excess of normal humanserum.

All eosinophils were stained with a selected anti-Eos L2 mAb, LS26-5H12.Neutrophils were not significantly stained by LS26-5H12 antibody underthe conditions of the assay. Based on the expected distribution of theEos L2 receptor, and that it functions in RANTES binding, MAb LS26-5H12appears to recognize the naturally expressed form of this receptor. Inaddition to the LS26-5H12 MAb, ˜five additional Mabs behaved similarly.

The LS26-5H12 hybridoma was further purified by limiting dilution. Inanother experiment, highly purified leukocyte subsets (purified asdescribed in Example 5) were stained with MAb LS26-5H12 and analyzed byflow cytometry (FIGS. 9A-9D). Staining profiles were representative ofat least 4 experiments. T Cells were identified based on CD3 staining.Monocytes and neutrophils were identified by forward and side scatter.

Highly purified eosinophils stained strongly with LS26-5H12 (FIG. 9A),suggesting abundant expression of the receptor on the surface ofeosinophils, and consistent with a high receptor number determined byligand binding and Scatchard analysis. Neutrophils, blood T cells, andmonocytes showed little or no staining with this MAb (FIGS. 9B-9D).These latter results, using antibody from the recloned hybridoma,suggest CKR-3 is selectively expressed on eosinophils, and is notappreciably expressed on other leukocyte types tested. However, it ispossible that a subset of T cells expresses the receptor.

Example 6 Selection of Stable L1.2 Cell Transfectants

2%-5% of transiently transfected COS, HEK-293 and CHO cells were surfacepositive as assessed using antibodies to FLAG-tagged receptor (seeabove), while substantial intracellular protein could be detected,suggesting inefficient protein trafficking. The L1.2 mouse pre-B cellline was used to select lines with higher levels of surface expression(see FIGS. 5A-5I) for further assessment of ligand binding specificityand signal transduction by CKR 3. This cell line has been usedsuccessfully for the study of other chemoattractant receptors (Honda,S., et al., J. Immunol., 152: 4026-4035 (1994)), and the expression oftransfected human chemokine receptors confers specific chemotacticability to various ligands (see below).

To monitor surface expression of CKR-3, a monoclonal antibody (MAb) wasproduced to the N-terminal region of the receptor, by immunizing micewith a synthetic peptide having a sequence corresponding to theN-terminal 35 amino acids of CKR-3. Anti-peptide MAbs were detected byELISA, and MAbs that recognize the native receptor were identified bytheir reactivity with human eosinophils, as well as their staining oftransient transfectants.

Construction of CKR-3/pcDNA3

PCR was used to modify the CKR-3 gene contained in the 1.8 kb genomicfragment by inserting a HindIII restriction site and optimal Kozaksequence immediately 5′ to the initiation codon. The coding region and448 bp of 3′ untranslated region were inserted into the HindIII site ofpcDNA3 (Invitrogen), placing the gene under the control of the human CMVimmediate early gene promoter of the vector. The details of theconstruction of this FLAG-tagged Eos L2 (CKR-3) receptor construct (alsoreferred to herein as CKR-3/pcDNA3) are provided in Example 3.

Transfection and Stable Cell Line Selection

The murine pre-B lymphoma cell line L1.2 was obtained from Dr. EugeneButcher (Stanford University) and maintained in RPMI-1640 supplementedwith 10% bovine serum. 20 μg of linearized, CKR-3/pcDNA3 was used totransfect the cell line as follows. L1.2 cells were washed twice in HBSSand resuspended in 0.8 ml of the same. The plasmid DNA was mixed withthe cells and incubated for 10 minutes at room temperature thentransferred to a 0.4 cm electroporation cuvette and a single pulseapplied at 250 V, 960 μF. The electroporation was followed by a 10minute incubation at room temperature. G418 was added to a finalconcentration of 0.8 mg/ml 48 hr post-transfection and the cells platedin 96 well plates at 25,000 cells/well. After 2-3 weeks under drugselection, G418 resistant cells were stained with 5H12 anti-receptor mAb(see below) and analyzed by FACScan®.

Lines with detectable surface staining were expanded and cloned severaltimes by limiting dilution. Clones with the brightest surface stainingwere further analyzed by Northern hybridization to confirm the presenceof transfected receptor as well as by RT-PCR using a T7 primercomplementary to the pcDNA3 vector as the 5′ primer and a CKR-3 specificprimer as the 3′ primer (not shown). No amplification was seen withoutaddition of reverse transcriptase.

For transient transfection, 20 μg of supercoiled DNA was used in theelectroporation exactly as described for stable cell line production.Cell surface staining was assessed after 48-72 hrs.

L1.2 cells transfected with CKR-3/pcDNA3 were diluted to 1×10⁶ cells/mlin tissue culture media. n-butyric acid (sodium salt, Sigma ChemicalCorp., Cat. No. B5887) was added to a final concentration of 5 mM(diluted from a 1M stock solution made in tissue culture media). Cellswere grown overnight (18-24 hours) at 37° C., 5% CO₂ prior to use. Lowerconcentrations have been used successfully (e.g., 2.5 mM and 1 mMn-butyric acid). n-butyrate treatment has been reported to induceprotein levels up to about 10-fold relative to uninduced controls (see,e.g., Palermo, D. P., et al., J. Biotech., 19: 35-48 (1991) andreferences cited therein). CKR-3 mRNA levels driven by the human CMVimmediate early gene promoter were elevated dramatically by n-butyratetreatment.

Monoclonal Antibody Production and Flow Cytometry

MAbs reactive with synthetic peptide were produced as described above inExample 5. MAbs were screened by ELISA as follows. 50 μl of peptide, ata concentration of 2 μg/ml in carbonate buffer, was used to coat NUNC96-well Maxisorp plates for at least 4 hours at 4° C. 300 μl/well ofblocking buffer (PBS+1% BSA) was added for at least 2 hours. Plates werewashed four times with PBS/Tween 20, and 50 μl of MAb supernatant wasadded to each well and incubated at 37° C. for one hour. Plates werewashed four times with PBS/Tween 20 and alkaline phospatase-conjugatedsecond antibody (Jackson ImmunoResearch Laboratories, West Grove Pa.)diluted 1:500 in PBS was added to each well. After an incubation at 37°C. for 30 minutes, plates were washed four times with PBS/Tween 20. Thesubstrate used for the color reaction was p-nitrophenylphosphatedissolved in diethanolamine buffer (Bio-Rad). Plates were read at 410 nmon an ELISA reader.

To determine which anti-peptide MAbs could recognize native, surfaceexpressed CKR-3, the anti-peptide MAbs were screened against transientlytransfected cells and eosinophils. For MAb staining, cells were washedonce with PBS, and resuspended in 100 μl PBS containing 2% FCS, 0.1%sodium azide (FACS buffer), 5 μg/ml purified antibody, 5 μg/ml MOPC-21IgG₁ isotype matched control MAb (Sigma) or 100 μl hybridoma culturesupernatant. After 30 min at 4° C., cells were washed twice in FACSbuffer, and resuspended in 100 μl of FITC-conjugated affinity purifiedF(ab′)₂ goat anti-mouse IgG (Jackson). After incubating for 30 minutesat 4° C., cells were washed twice in FACS buffer and analyzed byFACScan® to determine the level of surface expression. Propidium iodidewas used to exclude dead cells.

Surface Expression of Receptor on Stable Transfectants of the L1.2 CellLine

FIG. 10A shows detectable surface staining of the transientlytransfected receptor on a subpopulation of L1.2 cells, using ananti-receptor MAb, LS26-5H12. Untransfected L1.2 cells were negative(FIG. 10B). A stable cell line was constructed by limiting dilutioncloning of the transfectants and selection for higher surface stainingas described above. This process yielded lines that had much higherlevels of receptor expression (FIG. 10C). Northern blot analysisconfirmed the presence of transfected CKR-3 mRNA in one of thesubclones, designated E5, and its absence in untransfected L1.2 cells(not shown).

Example 7 Ligand Binding Specificity of Stable L1.2 Transfectants

Chemokines

Recombinant human chemokines were obtained from Peprotech, Inc. (RockyHill, N.J.), except for human eotaxin which was synthesized usingsolid-phase methods that were optimized and adapted to a fully automatedpeptide synthesizer (model 430A; Applied Biosystems, Inc., Foster City,Calif.) as described (Clark-Lewis, I., et al., Biochemistry, 30:3128-3135 (1991)). Human eotaxin is also commercially available fromPeprotech.

¹²⁵I-Labeling

¹²⁵I-labeled eotaxin was produced using the Bolton Hunter reagent (NEN),as described (Coligan, J. E., et al., Eds., 1992, Current Protocols inImmunology (New York: John Wiley and Sons)). The specific activity ofradiolabeled eotaxin was calculated to be 180 Ci/mM.

Ligand Binding

Chemokine binding to target cells was carried out using a modificationof a previously reported method (Van Riper, G., et al., J. Exp. Med.177: 851-856 (1993)). Cells were washed once in PBS and resuspended inbinding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSAand 0.05% azide) at a concentration of 1×10⁷/ml. Aliquots of 50 μl(5×10⁵ cells) were dispensed into microfuge tubes, followed by theaddition of cold competitor and radiolabeled chemokines. The finalreaction volume was 200 μl. Non-specific binding was determined byincubating cells with radiolabeled chemokines in the presence of 250-500nM of unlabeled chemokines. After 60 minutes incubation at roomtemperature, the cells were washed 3 times with 1 ml of binding buffercontaining 0.5 M NaCl. Cell pellets were then counted.

Competition is presented as the percentage of specific binding ascalculated by 100(S—B)/(T-B), where S is the radioactivity of thesample, B as background binding and T as total binding withoutcompetitors. Background binding was obtained by incubating cells withradiolabeled chemokine and at least 400-fold excess of unlabeledchemokines. The total binding of eotaxin to E5 cells was 11611±119 cpmand background binding 2248±745 cpm. The total binding of eotaxin toeosinophils was 7866±353 cpm and background binding 1148±518 cpm.Duplicates were used throughout the experiments and the standarddeviations were always less than 10% of the mean. All experiments wererepeated at least three times. Curve fit was calculated by KALEIDAGRAPHsoftware.

The E5 cell line described in Example 6 was tested for its ability tobind radiolabeled eotaxin. Cells were incubated with 0.6 nM ¹²⁵I-labeledeotaxin and various concentrations of cold competitor. FIG. 11A showsthat the transfected cells bound ¹²⁵I-labeled eotaxin specifically andwith high affinity. Scatchard analysis of the binding data indicated adissociation constant (Kd) of 1.5 nM (FIG. 11C), similar to the value of0.5 nM obtained using purified human eosinophils (FIG. 11D). Inaddition, both RANTES and MCP-3 were able to specifically compete forbinding. None of the other chemokines tested, including MIP-1α, MIP-1β,or IL-8 were able to specifically compete for radiolabeled ligand (FIG.12).

Chemotaxis Assays

Chemotaxis with human eosinophils was assessed using a modification of atransendothelial assay (Carr, M. W. et al., Proc. Natl. Acad. Sci. USA,91: 3652-3656 (1994)). The endothelial cells used for this assay werethe endothelial cell line ECV 304, obtained from the European Collectionof Animal Cell Cultures (Porton Down, U.K.). Endothelial cells werecultured on 6.5-mm diameter Biocoat® Transwell tissue culture inserts(Costar Corp., Cambridge Mass.) with a 3.0 μM pore size. Culture mediafor ECV 304 cells consisted of M199+10% Fetal Calf Serum, L-glutamine,and antibiotics.

Assay media consisted of equal parts RPMI 1640 and M199, with 0.5% BSA.24 hours before the assay, 2×10⁵ ECV 304 cells were plated onto eachinsert of the 24-well chemotaxis plate, and incubated at 37° C.Chemotactic factors (diluted in assay medium) were added to the 24-welltissue culture plates in a final volume of 600 μl. Endothelial-coatedtissue culture inserts were inserted into each well and 10⁶ cells wereadded to the top chamber in a final volume of 100 μl. The plate wasincubated at 37° C. in 5% CO₂/95% air for 4 hours.

The cells that had migrated to the bottom chamber were counted usingflow cytometry. 500 μl of the cell suspension from the lower chamber wasplaced in a tube, and relative cell counts were obtained by acquiringevents for a set time period of 30 seconds. This counting method wasfound to be reproducible, and enables gating on the leukocytes and theexclusion of debris or other cells. Counts obtained in this way matchclosely those obtained by counting with a microsope.

The same assay was used to assess chemotaxis of L1.2 cells or L1.2receptor transfectant cell lines, except that endothelial cells were notused to coat the Biocoat® Transwell tissue culture inserts.

CKR-3 Expression in L1.2 Cells Confers Chemotactic Responsiveness forEotaxin, RANTES and MCP-3

L1.2 receptor transfectants were tested for their ability to migrate inresponse to a panel of chemokines over a range of concentrations. TheCKR-3 expressing cell line E5 showed a chemotactic response to eotaxin,RANTES, and MCP-3 with a peak response to eotaxin at 100 ng/ml, althoughspecific migration could be detected as low as 10 ng/ml (FIG. 13A).While a response to RANTES was evident at both 10 ng/ml and 100 ng/ml,the magnitude of the response was not as great as with eotaxin. MCP-3appeared to be a less potent chemoattractant on the E5 cell line than oneosinophils, with no detectable migration below 100 ng/ml. Nosignificant response to other chemokines tested was seen with this cellline. In other control experiments, cells did not migrate to the bottomchamber when chemokine was added to the top well alone, confirming thatcell migration was chemotactic rather than chemokinetic (not shown).

The untransfected L1.2 cell line did not migrate in response to anychemokines tested (FIG. 13B). Indeed, a striking feature of the L1.2cell line was the very low background chemotaxis to non-specificligands. As a specificity control, L1.2 cells transfected with IL-8 RBmigrated specifically in response to IL-8 and GROα (FIG. 17C), as wellas NAP-2 and ENA-78 (not shown), but not to other CXC or CC chemokines.Other chemokine receptors which were introduced into L1.2 cells bytransfection also confer chemotactic ability to their specific ligands,including CKR-2 transfectants (which respond to MCP-1 and MCP-3), CKR-1transfectants (which respond to MIP-1α), and IL-8 RA transfectants(which respond to IL-8) (not shown). Pertussis toxin completelyabrogated the chemotactic response of both eosinophils and the CKR-3transfectants to eotaxin indicated that the receptor was signalingthrough the Gα subclass (Simon, M. I., et al., Science, 252: 802-808(1991)) in both normal and transfected cells (not shown).

The Chemotactic Profile of Eosinophils Resembles that of CKR-3Transfectants

In order to assess whether the function of normal eosinophils resembledthat of CKR-3 L1.2 transfectants, chemotaxis experiments were performedusing eosinophils from a number of normal individuals (humans), havinghigh levels of eosinophils (˜6 to 8% of WBC) (purified as described inExample 5). FIGS. 14A-14B show two characteristic patterns of eosinophilchemotaxis observed in two different individuals. One pattern wascharacterized by a robust migration to eotaxin, and a lesser response toRANTES and MCP-3 (FIG. 14A). The other pattern showed essentiallyequivalent chemotaxis in response to eotaxin, RANTES and MCP-3 (FIG.14B). These patterns were not due to variations in the assay, sincewithin each individual, they were highly reproducible over a long periodof time. MIP-1α showed only weak chemotactic activity for eosinophils inthe second class of individuals.

Example 8 Cloning of a cDNA Encoding Eos L2

Construction of an Eosinophil cDNA Library

Eosinophils were obtained from a patient (M.V.) diagnosed withidiopathic hyper-eosinophilic syndrome (Costa, J. J. et al., J. Clin.Invest., 91: 2673 (1993). RNA was isolated using a standard guanidiniumisothiocyanate/cesium chloride method (In: Current Protocols InMolecular Biology, Vol. 1, Ausubel, F. M. et al., Eds., (John Wiley &Sons: New York, N.Y.) page 4.2.2-4.2.3 (1991)). mRNA was obtained usingDynabeads® (Dynal, Inc.), and the bacteriophage library was constructedusing the SUPERSCRIPT™ Lambda System for cDNA Synthesis and λ Cloning(Gibco BRL, Life Technologies) which comes with λgt22A, NotI-SalI arms.

Library Screening

We screened approximately 750,000 bacteriophage plaques of the resultinghuman eosinophil cDNA library in duplicate. The probe used was a fulllength radiolabeled cDNA probe (p4 cDNA) which encodes the MIP-1α/RANTESreceptor (CKR-1)(Gao et al., J. Exp. Med., 177:1421 (1993)). The p4 cDNAwas cloned into the BamHI (5′) and XhoI (3′) sites of pcDNAI(Invitrogen). A BamHI-XhoI fragment of this clone (i.e., p4 cDNA inpcDNAI) was obtained by restriction digestion, and isolated using GeneClean (Bio101). The fragment was labeled with ³²P using a random primerlabeling kit (Boehringer Mannheim Biochemicals).

Filters were prehybridized by incubation for two hours at 42° C., in asolution of 50% formamide, 5×SSC, 1× Denhardt's, 10% Dextran Sulfate, 20mM TRIS, pH 7.5, 0.1% SDS (sodium dodecyl sulfate). Hybridization wasperformed overnight at 42° C. in the same solution. Eosinophil cDNAlibrary filters were then washed two times with 2×SSC/0.1% SDS at roomtemperature, and two times with 2×SSC/0.1% SDS at 42° C. Each wash wasfor 30 minutes. Filters were exposed overnight and positive plaques werepicked in duplicate. Clones were further evaluated when positive induplicate after the low stringency washes.

Characterization of cDNA Clones

Plaques were plaque purified, and DNA was isolated by a small scalephage lysis protocol (In: Current Protocols In Molecular Biology, Vol.1, Suppl. 10, Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York,N.Y.), page 1.13.7 (1991). The bacteriophage DNA was digested with EcoRI(site in arm of vector) and NotI. The inserts released by digestion werevisualized on a gel, and were found to be approximately 1.6 kb inlength. The ˜1.6 kb insert present in a plaque designated Mip-16 orM-16, was isolated using Gene Clean (Bio101), and was cloned into theEcoRI and NotI sites of Bluescript® vector KS (Stratagene), which hadbeen digested with both EcoRI and NotI to produce asymmetric ends. Theligated plasmid was introducted into XL1-Blue E. coli cells (Stratagene)made competent as described by Hanahan (Hanahan, D., (1985), In: DNACloning, Volume 1, D. M. Glover, Ed. (IRL Press: Washington, D.C.), pp.109-135).

Dideoxy sequencing of the M-16/Bluescript construct was performed usinga dideoxynucleotide sequencing kit obtained from USB (United StatesBiochemical, Cleveland, Ohio). The nucleotide sequence of this clone wasdetermined to encode a novel protein with a high degree of homology tothe MIP-1α/RANTES receptor; however, from the sequence data, the clonedid not appear to be full-length.

In order to identify a full-length clone, 15-20 additional plaques wereisolated and purified, and the inserts present in the phage werecharacterized by restriction enzyme analysis and/or sequencing. Anotherλ clone, designated M31, which was isolated was found to contain a ˜1.8kb insert. The insert was cloned into the EcoRI and NotI sites ofBluescript® vector KS (Stratagene), and introduced into XL1-Blue E. colicells (Stratagene) as described above. DNA sequencing of this clone (M31insert in Bluescript, referred to as M31/Bluescript construct) wasperformed as described above, and revealed that it encoded a full-lengthreceptor.

The M31 insert was released from the M31/Bluescript construct bydigestion with EcoRI and NotI. The resulting fragment was isolated usingGene Clean (Bio101), and was inserted into the EcoRI and NotI sites ofvector Ap^(r)M9, which had been digested with both EcoRI and NotI toproduce asymmetric ends. Vector Ap^(r)M9 (de Fougerolles, A. R. et al.,J. Exp. Med., 177: 1187-1192 (1993)) is a derivative of CDM8(Invitrogen) containing the β-lactamase from pBluescript and apolylinker from pSP64. The resulting construct, designated A31, wasintroduced into competent XL1-Blue cells.

The nucleotide sequence of the full-length cDNA and the predicted aminoacid sequence of the encoded protein are shown in FIGS. 2A-2C (see alsoSEQ ID NO:3 and SEQ ID NO:4). The cDNA sequence shown in FIGS. 2A-2C wasdetermined from clones A31 (bases 15-365 (numbering as in FIGS. 2A-2C)),and the M-16/Bluescript construct (bases 366 to 1152 (numbering as inFIGS. 2A-2C)). A comparison of the amino acid sequence of the novelreceptor with other proteins revealed that the novel receptor and theMIP-1α/RANTES receptor share 62% sequence identity, and the novelreceptor and the MCP-1 receptor share 50.57% sequence identity. Sequenceidentity was determined using the Wisconsin UW GCG package (programgap), with the Needleman and Wunsch algorithm (Needleman and Wunsch, J.Mol. Biol. 48:443-453 (1970)).

Northern Analysis

RNA for Northern analysis was obtained from a patient havinghyper-eosinophilia. The eosinophils were isolated as described (Costa,J. J., et al., J. Clin. Invest., 91: 2673 (1993)). Total eosinophil RNAwas isolated using standard procedures (In: Current Protocols InMolecular Biology, Vol. 1, Ausubel, F. M. et al., Eds., (John Wiley &Sons: New York, N.Y.) page 4.2.2-4.2.3 (1991)). The total RNA wasfractionated on a 1% agarose gel, and then blotted onto GeneScreenfilters (New England Nuclear). Filters were probed at high stringencyaccording to the manufacturer's protocol for high stringency washing ofGene Screen blots (New England Nuclear).

Several Northerns were prepared. One involved probing with theEcoRI-NotI fragment of the M16/Bluescript construct, and others wereprobed with the EcoRI-NotI fragment from clone A31. Both EcoRI-NotIfragments include the 3′ untranslated regions. Probes were labeled with³²P using a random primer labeling kit (Boehringer MannheimBiochemicals).

The Northern blots each revealed a very strong signal of approximately1.8 kb in total human eosinophil RNA. This result indicates that the A31RNA is expressed at very high levels in eosinophils from this patient.

Example 9 Expression of cDNA Encoding Eos L2 Receptor and Ligand BindingStudies

Constructs

Vectors A31 (described above) and A31-pcDNA3 were used for expressionand binding analyses. To construct A31-pcDNA3, vector A31 was digestedwith EcoRI and NotI, the ˜1.8 kb insert was isolated using Gene Clean(Bio101), and was inserted into the EcoRI and NotI sites of vectorpcDNA-3 (Invitrogen), which had been digested with both EcoRI and NotI.The ligated construct, designated A31-pcDNA3, was introduced intocompetent XL1-Blue cells.

Transient Transfections

Transient transfections using A31 in the kidney cell line 293 initiallysuggested high affinity binding of A31 with radioactive RANTES. Theseinitial binding studies have been difficult to reproduce. Accordingly,stable cell lines have subsequently been produced with A31/pcDNA3 stablyintegrated into both RBL (rat basophilic leukemia) and 293 cells. RBLcells (Accession No. ATCC CRL 1378) were obtained from the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, and 293 cells (Accession No. ATCC CRL 1573) were a gift fromI. Charo, Gladstone Cardiovascular Institute.

Stable Cell Lines

Stable cell lines were constructed as follows. A31 pcDNA3 was linearizedby digestion with NotI. The linearized plasmid was introduced into RBLand 293 cells by electroporation. Confluent 293 and RBL cells growing in100×20 mm plates were trypsinized, resuspended in 1 cc of phosphatebuffered saline (PBS) and electroporated in a 0.4 cm cuvette (BioRad)with settings of 960 microfarads and 250 volts. Stable transfectantswere isolated by positive selection in medium containing geneticin.Specifically, the cells were first cultured in DMEM (BRL), 10% fetalcalf serum for several days, and then were switched to DMEM, 10% fetalcalf serum with 0.9 mg/cc of Geneticin (BRL). (DMEM, Dulbecco's ModifiedEagle's Medium). After 3 weeks, surviving colonies were isolatedsterilely with cloning cylinders, and individual clones were grown inindividual wells in DMEM, 10% fetal calf serum with 0.9 mg/cc ofGeneticin (BRL).

Surviving clones which expressed A31 RNA at high levels were detected byNorthern analysis. 120 stable transfectants of the RBL line, and 38stable transfectants of the 293 cell line, were screened. Specifically,RNA from individual clones was isolated using the acid phenol method(Chomczynski, P. and N. Sacchi, Anal. Biochem., 162: 156-159 (1987)).RNA was fractionated by electrophoresis, blotted onto GeneScreen (NewEngland Nuclear), and Northern blots were probed according to themanufacturer's suggestion for high stringency wash. The EcoRI-NotIinsert from plasmid A31 was isolated, radiolabeled with ³²P using therandom primer labeling kit (Boehringer Mannheim Biochemicals), and usedas a probe. RNA was quantified by ethidium bromide staining on gels.Untransfected 293 or RBL cells were used as negative controls for thecorresponding transfectants.

Stable cell lines designated A31-293-#8, A31-293 #9, A31-293-#17, andA31-293-#20 were subsequently found to express A31 RNA at very highlevels relative to other lines. Clone A31-293-#20 which highly expressesthe A31 message by Northern analysis, was selected for further study.

One RBL line was found to express low-medium amounts of RNA, but did notappear to bind RANTES under the conditions used (not shown).

Ligand Binding

Stable clone A31-293-#20 was grown in quantities sufficient for bindingassays. In particular, cells were grown in 100 mm plates in DMEM, 10%fetal calf serum, 0.9 mg/cc geneticin. Plates were grown to confluence,and membranes were prepared as follows. Culture medium was removed, andthe cells were washed with phosphate buffered saline. Cells wereharvested by washing with TEN (40 mM TRIS, pH 7.5, 1 mM EDTA, and 150 mMNaCl). The cells were frozen in liquid nitrogen, thawed at roomtemperature, and the membrane fraction was collected by centrifugationin a conical tube for 10 minutes at 18,000 rpm. Each binding point wasdetermined using one-half of the membranes harvested from a single 100mm plate grown to confluence.

¹²⁵I-labeled RANTES was purchased from New England Nuclear, and coldRANTES was purchased from Peprotech (Princeton, N.J.). ¹²⁵I-labeledMCP-3 was a gift from New England Nuclear, and cold MCP-3 was a giftfrom J. Van Damme, Rega Institute for Medical Research, University ofLeuven, B-3000 Leuven, Belgium (see also, Opdenakker, G. et al.,Biochem. Biophys. Res. Commun., 191(2): 535-542 (1993)). Binding assayswere performed as described by Van Riper, G. et al., J. Exp. Med., 177:851 (1993), with the following modifications. In particular, the bindingto membranes of 0.125 nanomolar of ¹²⁵I-RANTES was performed in thepresence of varying concentrations of unlabeled ligand. Binding bufferwas 50 mM Hepes, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA, pH 7.2. Radiolabeledand cold ligand were added simultaneously to the membranes (see above),and incubated for 1.5 hours at room temperature. The binding reactionwas added to 2 cc of wash buffer (0.5 M NaCl, 50 mM Hepes, 1 mM CaCl₂, 5mM MgCl₂, 0.5% BSA, pH 7.2), mixed by vortexing, and then placed onpolyethyleneimine-treated Whatman GFC filters. Filters were washed withan additional two ccs of wash buffer. Activity retained on filters afterwashing was determined by scintillation counting. Filters were placed in5 cc of scintillation fluid and were then counted in a miniaxi-betaliquid scintillation counter (United Technologies, Packard, DownersGrove, Ill.). All points were determined in triplicate, except for thepoint at 2 nM, which was determined in duplicate.

The results of the assay indicated high affinity binding of RANTES tothe receptor encoded by the A31 clone (FIG. 15). Scatchard analysis ofthe data indicated a K_(d) of ˜2.5 nM for RANTES, which is what isexpected in normal cells.

Binding of MCP-3 to membranes from clone A31-293-#20 was also assessedusing the ligand binding assay described above for RANTES binding toA31-293-#20 membranes (FIG. 16). Binding reactions contained 0.125nanomolab ¹²⁵I-labeled MCP-3.

In addition, specificity of binding was assessed by determining theextent to which labeled MCP-3 (bound in the absence of cold MCP-3),could be displaced by cold MCP-3 (FIG. 17). All points were taken induplicate.

The MCP-3 bound to membranes from untransfected cells could not bedisplaced by ¹²⁵I-labeled MCP-3, indicating non-specific binding. Incomparison, the MCP-3 bound to membranes from A31-293-#20 cells could bedisplaced by hot MCP-3, which is indicative of specific binding.

The results of these assays indicate that the receptor encoded by theA31 cDNA specifically binds human MCP-3.

Example 10 Human Eosinophils Respond to Numerous cc Chemokines ThroughOne Predominant Receptor

Cells, Cell Lines, and Tissue Culture. Eosinophils were isolated fromheparinized blood using CD 16 microbeads (Miltenyi Biotec, Auburn,Calif.), as described in Ponath, P. D., et al., J. Clin. Invest., 97:604612 (1996) and were shown cytologically to be ≧99% pure. Neutrophils andPBMCs were isolated as described in Ponath, P. D., et al., J. Clin.Invest., 97:604 612 (1996). To generate CD3 blasts, 2×10⁶ PBMC/ml inRPMI 1640 plus 10% FCS were added to tissue culture plates first coatedwith the anti-CD3 antibody TR66. After 4-6 days blasts were removed tofresh media and supplemented with IL-2 (provided by AntonioLanzavecchia, Basel) at 50 units/ml. Other cell lines used includedtransfectants of the L1.2 murine pre B cell lymphoma, expressing highlevels of either CCR3 (see below; Ponath, P. D., et al., J. Exp. Med.,183:2437 2448 (1996)), IL-8 RA (Ponath, P. D., et al., J. Exp. Med.,183:2437-2448 (1996)), IL-8 RB (Ponath, P. D., et al., J. Exp. Med.,183:2437-2448 (1996)), CCR2b, CCR4 and CCR5, and CCR1 (Campbell, J. J.,et al., J. Cell Biol., 134:255-266 (1996)). Transfectants weremaintained in RPMI 1640 supplemented with 10% bovine serum and 800 μg/mlG418. The different transfectants were monitored for expression of therelevant receptors, using mAbs specific for CCR3 (Ponath, P. D., et al.,J. Exp. Med., 183:2437-2448 (1996)), IL-8 RA, IL-8 RB, or CCR2 (Qin, S.,et al., Eur. J. Immunol. 26:640-647 (1996); (Ponath, P. D., et al., J.Clin. Invest., 97:604-612 (1996)). For CCR4 and CCR5, expression wasmonitored using the anti-flag mAb M2, since these receptors wereconstructed with this epitope at the N-terminus.

Human eosinophils were cultured in RPMI 1640 with 10% FCS and 5 ng/ml ofrecombinant human IL-5 (Genzyme Corp., Cambridge, Mass.), for 5-7 days,using tissue culture flasks containing subconfluent monolayers of ECV304cells.

MAbs to IL-8 RA, IL-8 RB, and CCR2 (MCP-IR) have been described (Qin,S., et al., Eur. J. Immunol. 26:640-647 (1996)). mAb staining of cellswas performed using standard procedures, as described previously(Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)). Toenumerate antibody binding sites per cell, the F/P ratio of 7B11-FITCwas determined with Simply Cellular beads (Flow Cytometry StandardsCorp., San Juan, PR) and the FACScan® was calibrated with Quantum 26beads (Flow Cytometry Standards Corp.), according to the manufacturer'sinstructions. 100 μl of whole blood from donors was reacted with asupersaturating amount (400 ng) of 7B11 -FITC in PBS with 0.5% azide.Red cells were lysed with ammonium chloride lysing solution and the meanchannel fluorescence of 7B11 stained cells was determined by flowcytometry.

Expression Vector Construction and Generation of CCR3 StableTransfectants

The 1.8 kb CKR-3 (CCR3) genomic fragment, which was ligated into theHindIII site of the pBluescript II KS+ vector (Stratagene) (Example 2),was modified for expression by insertion of a HindIII restriction siteand optimal Kozak sequence immediately 5′ to the initiation codon in afour-stage process as described in Example 3 (Construction ofFLAG-tagged Eos L2 (CKR 3) Receptor Construct).

The murine pre-B lymphoma cell line L1.2 was maintained in RPMI-1640supplemented with 10% bovine serum. 20 μg of the FLAG-taggedCKR-3/pcDNA3 construct (Example 3) were linearized by digestion withScaI and used to transfect the L1.2 cell line as follows. L1.2 cellswere washed twice in HBSS and resuspended in 0.8 ml of the same buffer.The plasmid DNA was mixed with the cells and incubated for 10 minutes atroom temperature, transferred to a 0.4-cm electroporation cuvette, and asingle pulse was applied at 250 V, 960 μF. The electroporation wasfollowed by a 10 minute incubation at room temperature. G418 was addedto a final concentration of 0.8 mg/ml 48 hours after transfection andthe cells were plated in 96-well plates at 25,000 cells/well. After 2-3weeks under drug selection, G418-resistant cells were stained with 5H12anti-receptor monoclonal antibody, and analyzed by FACScan® (BectonDickinson & Co., Mountain View, Calif.). For mAb staining, cells werewashed once with PBS, and resuspended in 100 μl PBS containing 2% FCS,0.1% sodium azide (FACS® buffer), 5 μg/ml affinity purified antibody or5 μg/ml MOPC-21 IgG₁-isotype matched control mAb (Sigma Chemical Co.,St. Louis, Mo.), or 100 μL hybridoma culture supernatant. 5H12 antibodywas used as hybridoma culture supernatant. After 30 minutes at 4° C.,cells were washed twice with FACS® buffer, and resuspended in 100 μlFITC-conjugated, affinity-purified F(ab′)₂ goat anti-mouse IgG (JacksonImmunoResearch Laboratories). After incubation for 30 minutes at 4° C.,cells were washed twice in FACS® buffer and analyzed by FACScan®.Propidium iodide was used to exclude dead cells. Stable transfectantswere treated with 5nM n-butyric acid (Sigma Chemical Co., St. Louis,Mo., Catalog No. B 5887) 24 hours prior to analysis (FACS staining orbinding) or immunization. All stable transfectants, including the L1.2transfectants described in FIGS. 18A and 18C were treated with n-butyricacid. Lines with detectable surface staining were expanded and clonedseveral times by limiting dilution. As a negative control, CKR-3transfected cells were stained with an irrelevant control IgG1 MAb(MOPC-21) and the same second antibody. In addition, control L1.2 cellstransfected with IL-8RB, which were processed in parallel, were stainedwith 5H12 and second antibody. A CKR-3 transfected clone having thebrightest surface staining as assessed by fluorescence intensity wasused as immunogen as described below. Generally, the mean channelfluorescence intensity of the 5H12-stained cell preparation was 2-3 logshigher than staining of the controls. The transfectants used in theimmunization which yielded the monoclonal antibody designated 7B11,displayed a fluorescence intensity two logs higher that theMOPC-21-stained and the IL-8RB controls.

Clones with the brightest surface staining were further analyzed byNorthern hybridization to confirm the expression of transfected receptoras well as by RT-PCR using a T7 primer complementary to the pcDNA3vector as the 5′ primer and a CKR-3-specific primer as the 3′ primer. Noamplification was seen without addition of reverse transcriptase.

Monoclonal Antibody Production and Flow Cytometry

L1.2 CCR3 transfected cells prepared as described above were washedthree times in PBS and resuspended in 200 μl PBS/10⁷ cells. Monoclonalantibodies reactive with CCR3 were generated by immunizing C57BL6 micewith 10⁷ L1.2 CCR3 transfected cells, intraperitoneally, five to sixtimes at 2 week intervals. The final immunization was injectedintravenously. Four days later, the spleen was removed and cells werefused with the SP2/0 cell line as described (Coligan, J. E. et al.,1992, In: Current Protocols In Immunology (John Wiley and Sons, NewYork), Unit 2.5.4).

Monoclonal antibodies reactive with CCR3 were identified usinguntransfected and CCR3 transfected L1.2 cells, and immunofluorescentstaining analysis using a FACScan® (Becton Dickinison & Co., MountainView, Calif.). Hybridoma culture supernatants were used in an indirectimmunofluorescence assay in a 96-well format using anti-mouse Ig-FITC.Untransfected and CCR3 transfected L1.2 cells were washed once with PBS,and resuspended in 50 μl PBS containing 2% FCS, 0.1% sodium azide (FACS®buffer). 50 μL hybridoma culture supernatant was added. After 30 minutesat 4° C., cells were washed twice with FACS® buffer, and resuspended in100 μl FITC-conjugated, affinity-purified F(ab′)₂ goat anti-mouse IgG(Jackson ImmunoResearch Laboratories). After incubation for 30 minutesat 4° C., cells were washed twice in FACS® buffer and analyzed byFACScan®. Antibodies which stained CCR-3 transfectants but notuntransfected L1.2 cells were selected. Two monoclonal antibodiesreactive with CCR3 were obtained from two different fusions. One ofthese antibodies, produced by the 7B11 hybridoma, was designated 7B11.

Chemokines, Chemotaxis Assays, and Ligand binding Assay. Recombinanthuman chemokines were obtained from Peprotech (Rocky Hill, N.J.), exceptfor eotaxin, described previously (Ponath, P. D., et al., J. Clin.Invest., 97:604 612 (1996)), which was a gift of Dr. Ian Clark Lewis.Chemotaxis of human eosinophils was assessed using a modification of atransendothelial assay (Carr, M. W., et al., Proc. Nat'l. Acad. Sci.USA, 91:3652 3656 (1994)), using the cell line ECV304 as described(Ponath, P. D., et al., J. Clin. Invest., 97:604 612 (1996). Cells thathad migrated to the bottom chamber were placed in a tube, and relativecell counts were obtained using the FACScan.

¹²⁵I-labeled eotaxin was obtained from Amersham (Arlington Heights,Ill.), and its specific activity was stated to be 2000 Ci/mM. Chemokinebinding to target cells was carried out as described previously (Ponath,P. D., et al., J. Clin. Invest., 97:604 612 (1996); Van Riper, G., etal., J. Exp. Med., 177: 851-856 (1993)). Duplicates were used throughoutthe experiments and the standard deviations were always <10% of themean. All experiments were repeated at least three times. Curve fit andconcentrations that inhibit 50% specific binding (IC50) were calculatedby KALEIDAGRAPH software (Synergy Software, Reading, Pa.).

Measurement of intracellular calcium concentration ([Ca²⁺]_(i)). 50 μgFura-2 AM (Molecular Probes, Eugene Oreg.) was dissolved in 44 μl ofDMSO, and this was diluted to 4.4 ml with loading buffer (Hanks BalancedSalt Solution, Gibco/BRL, catalogue # 14025-092 containing 2% BSA).Eosinophils were resuspended in loading buffer at 10⁷ cells/ml, and 1.5ml of cells was mixed with 300 μl of the Fura-2 solution for 30 minutesat 37° C. Following labeling, excess dye was removed by centrifugationand cells were resuspended at a concentration of 10⁶/ml in 125 mM NaCl,5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 0.5 mM glucose, 0.025% BSA and 20 mMHEPES, pH 7.4. [Ca²⁺]_(i) was measured using excitation at 340 and 380nm on a Hitachi F-2000 fluorescence spectrometer. Calibration wasperformed using 1% NP-40 for total release and 25 μM EGTA to chelatefree Ca²⁺.

Results

Complete blocking of eotaxin, RANTES and MCP-3 binding to CCR3transfectants using a mAb, 7B11. L1.2 transfectants expressing highlevels of CCR3 were selected using the anti-CCR3 peptide mAb 5H12(Example 5, also referred to herein as LS26-5H12; Ponath, P. D., et al.,J. Clin. Invest., 97:604-612 (1996)). mAbs were produced to surfaceexpressed CCR3 and one mAb, 7B11, was identified that reacted with L1.2cells transfected with CCR3, but not with L1.2 cells transfected withCCR1, CCR2b, CCR4, CCR5, CXCR1, or CXCR2 (FIG. 18A). mAb 7B11 stainedhuman eosinophils intensely (FIG. 18B). This mAb was unreactive withlymphocytes, CD3 activated T cells, and monocytes. Staining onneutrophils was largely negative, 10 although a small percentage ofthese cells may express very low levels of the receptor.

The small subset of granulocytes stained intensely by 7B11 (FIG. 18A)were eosinophils which were contained in the granulocyte gate.

mAb 7B11 was assessed for its ability to inhibit ¹²⁵I-labeled eotaxin,¹²⁵I-RANTES, ¹²⁵I-MCP-2 and ¹²⁵I-MCP-3 binding to CCR3 transfectants.mAb 7B11 inhibited binding completely of ¹²⁵I-labeled eotaxin to thetransfectants (FIG. 18C), and this inhibition was as efficient as thatobtained with 100 nM cold eotaxin. This indicated that mAb 7B11 was ableto completely block eotaxin binding to CCR3. This mAb also completelyinhibited ¹²⁵I-labeled RANTES, ¹²⁵I-labeled MCP-3 and ¹²⁵I-labeled MCP-2binding to CCR3 transfectants (FIG. 18C), indicating that the epitoperecognized by 7B11 was involved in the binding of numerous CCchemokines. In contrast, mAb 7B11 failed to inhibit RANTES binding toCCR1 transfectants (FIG. 18C).

mAb 7B11 blocks binding of radiolabeled eotaxin, RANTES and MCP-3 toeosinophils. To test if eotaxin, RANTES and MCP-3 binding to eosinophilswas occurring through CCR3, binding of radiolabeled chemokines toeosinophils was performed in the presence of various concentrations ofthe blocking mAb 7B11, or a control mAb (FIG. 19). ¹²⁵I-labeled eotaxinbinding to eosinophils could be completely inhibited using anappropriate amount of 7B11 mAb, consistent with results indicating thateotaxin binds only to CCR3 on eosinophils (Ponath, P. D., et al., J.Exp. Med., 183:2437-2448 (1996)). However RANTES and MCP-3 are known tobind chemokine receptors in addition to CCR3 (Neote, K., et al., Cell72:415-425 (1993); Gao, J. L., et al., J. Exp. Med., 177:1421-1427(1993); Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)). FIG.19 shows that mAb 7B11 also inhibited ¹²⁵I-labeled RANTES and¹²⁵I-labeled MCP-3 binding to eosinophils. 50 ng/ml of mAb 7B11 wassufficient to achieve complete blockade of all chemokine binding tonormal eosinophils, similar to the inhibition achieved with 2500-foldexcess of cold chemokines. Slightly lower amounts of mAb 7B11 wererequired to block RANTES and MCP-3 binding, which is consistent with thelower affinity of RANTES and MCP-3 for CCR3 (Ponath, P. D., et al., J.Exp. Med., 183:2437-2448 (1996)).

Inhibition of eosinophil chemotaxis to CC chemokines using anti CCR3mAb. Chemotaxis experiments were performed using eosinophils from normalindividuals with moderately high levels of eosinophils (˜3 to 6% ofWBC). FIG. 20A shows that mAb 7B11 was able to inhibit completely thechemotaxis of eosinophils to eotaxin in a dose dependent manner. 5 10ug/ml was required to achieve 100% inhibition, using 100 ng/ml (12.5 nM)of chemokine in the bottom well. FIG. 20B shows that the eosinophilchemotactic responses to RANTES, MCP-2, MCP-3, and MCP-4 could beinhibited totally using 5 10 ug/ml of mAb 7B11. 7B11 was unable toinhibit eosinophil chemotaxis to C5a (FIG. 20B). Moreover, mAb 7B11 wasunable to inhibit PBMC chemotaxis to RANTES, which occurs throughchemokine receptors other than CCR3. Donor to donor variation ineosinophil chemotactic responses to chemokines has been observed(Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)). However, inall individuals examined thus far (n=8), mAb 7B11 was able to inhibitby >95% the migration of eosinophils to eotaxin, RANTES, MCP-2, MCP-3,and MCP-4.

mAb 7B11 inhibits changes in [Ca²⁺]i by eosinophils in response to CCchemokines. Eotaxin, RANTES, MCP 2, MCP-3 and MCP-4 induce changes in[Ca²⁺]i by human eosinophils (Ponath, P. D., et al., J. Clin. Invest.,97:604-612 (1996); (Uguccioni, M., et al., J. Exp. Med., 183:2379-2384(1996)). To examine the agonist/antagonist function of mAb 7B11,eosinophils were assessed for [Ca²⁺]i following injection of mAb 7B11,or an irrelevant control mAb. Eosinophils incubated with the irrelevantmAb still produced changes in [Ca²⁺]i following injection of optimalamounts of eotaxin, RANTES, MCP-2, MCP-3 and MCP-4 (FIGS. 21A, 21C, 21E,21G and 21I). C5a, a potent stimulator of eosinophil [Ca²⁺]i, was usedas a control.

Eosinophils incubated with 6.4 μg/ml of 7B11 mAb for 40 seconds wereunable to respond to eotaxin, RANTES, MCP 2, MCP-3 and MCP-4 (FIGS. 21B,21D, 21F, 21H and 21J). This inhibition was not due to receptormodulation from the cell surface, since this effect was rapid, andimmunofluorescent staining of eosinophils incubated with mAb 7B11 atroom temperature revealed intense staining. In addition, mAb 7B11 wasantagonistic rather than agonistic, since concentrations as high as 10αg/ml of mAb failed to induce a change in [Ca²⁺ 9 i. 7B11 treatedeosinophils showed changes in [Ca²⁺]i to C5a (FIG. 21). mAb 7B11 had noeffect on the [Ca²⁺]i of butyrate differentiated HL 60 cells to MIP-1αor RANTES, a response that is mediated through receptors other thanCCR3.

IL-5 primed eosinophils respond to CC chemokines through CCR3 butupregulate IL-8 receptors. Eosinophils from eosinophilic individuals,and normal eosinophils primed in vitro with IL-5, respond to IL-8 inchemotaxis assays (Schweizer, R. C., et al., Blood, 83:3697 3704 (1994);Sehmi, R., et al., Clin. Exp. Allergy, 23:1027-1034 (1994)), suggestingthat activated eosinophils have altered chemokine receptor expression.To test whether primed or activated eosinophils respond to CC chemokinesin the same manner as do normal eosinophils, blocking experimentssimilar to those shown in FIGS. 20 and 21A-21J were performed using day5 to 7 IL-5 stimulated eosinophils, and eosinophils from an eosinophilicindividual. The IL-8 receptors, CXCR1 and CXCR2, were undetectable bymAb staining on eosinophils from all normal individuals examined (n=12)(FIG. 22A). However following 5-7 days culture in vitro with human IL-5,CXCR2 and (to a lesser degree) CXCR1 were detectable on the surface ofeosinophils, as detected using anti CXCR2 mAbs and flow cytometry (FIG.22B), and this expression paralleled the ability of these eosinophils tomigrate to IL-8 in chemotaxis assays (not shown). In the oneeosinophilic donor examined (18-25% of WBC were eosinophils, for >1year), CXCR2 was expressed on eosinophils at a slightly lower level(FIG. 22C).

mAb 7B11 was still able to block completely the calcium responses ofboth IL-5 primed eosinophils (FIG. 22D), and eosinophils from theeosinophilic donor, to eotaxin and RANTES (FIG. 22D), as well as MCP-2,MCP-3, and MCP-4, in a similar fashion to that described for normaleosinophils. mAb 7B11 had no effect on IL-8 responses (FIG. 22D), andMIP-1α responses were not evident in these experiments. CCR3 expressionwas assessed on the IL-5 primed eosinophils, and from eosinophils fromnumerous healthy individuals. The number of 7B11 binding sites pereosinophil from healthy individuals was calculated to be 17,400±1600(n=12), and no significant differences were observed following IL-5stimulation. However in the one eosinophilic donor analyzed, the numberof 7B11 binding sites was found to be 26,000.

Discussion

The functional effects of all of the efficacious chemokines foreosinophils characterized, including eotaxin, RANTES, MCP-2, MCP-3, andMCP-4, could be blocked completely with an anti-CCR3 mAb with potentantagonistic activity. This mAb was specific for CCR3, and no inhibitoryeffects on other chemoattractant receptors were observed. These resultsfurther establish that CCR3 is the principal receptor for eosinophilresponses to CC chemokines, and questions an essential role for CCR1,CCR2, CCR4 or CCR5.

The predominant CC chemokine receptor on eosinophils is CCR3. Thisreceptor is expressed at a high level, as shown by ligand bindingstudies and mAb staining. A recent study suggested that humaneosinophils express MIP-1α receptors, either CCR1, CCR4 or CCR5, atabout 1-5% of the levels of CCR3 (Daugherty, B. L., et al., J. Exp.Med., 183:2349-2354 (1996)), and modest eosinophil chemotactic responsestowards MIP-1α a have been observed in some individuals (see above;Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996); Ponath, P.D., et al., J. Clin. Invest., 97:604-612 (1996)). However, the resultsusing MAB 7B11 indicate MIP-1α receptor(s) contribute little to thefunctional responses of eosinophils to RANTES or MCP-3. Donor variationwas observed in eosinophil responses to the CC chemokines, however theseresponses were blocked completely in all individuals, using mAb 7B11,indicating that if other CC chemokine receptors are present, they have aminor functional significance. In addition, responses of IL-5-stimulatedeosinophils to CC chemokines could also be blocked by mAb 7B11,suggesting that no new receptors were upregulated on cytokine primedeosinophils, as happens for IL-2 primed T cells (Loetscher, P., et al.,J. Exp. Med., 184:569-577 (1996)). The relevance of IL-8 receptors onIL-5 primed or activated eosinophils is uncertain. The phenotypic andfunctional analyses described herein are consistent with previousreports showing that IL-5 stimulated eosinophils, or eosinophils fromeosinophilic donors, respond to IL-8 in chemotaxis assays (Schweizer, R.C., et al., Blood, 83:3697-3704 (1994); Sehmi, R., et al., Clin. Exp.Allergy, 23:1027-1034 (1994)).

Thus, as described herein, a fully antagonistic mAb to a CC chemokinereceptor has been identified. A CCR3 antagonist has application in thetreatment of diseases such as asthma, where an inhibition of eosinophilmigration to the airways is beneficial. The role of Eotaxin CCR3 ineosinophil migration to the airways in asthma is suggested since aselective recruitment of eosinophils often occurs in this disease.Moreover, eotaxin and other chemokines are highly upregulated in theairways of asthma patients (J. Rottmann and D. Ringler), as well as inanimal models of allergic airway disease (Jose, P. J., et al., J. Exp.Med., 179:881-887 (1994); Gonzalo, J. A., et al., Immunity, 4:1 14(1996)).

Example 11 Blockade of Eosinophil Degranulation Induced by Eotaxin,RANTES and MCP-3 by Anti-CCR3 Monoclonal Antibody 7B11. Effect ofAnti-CCR3 Monoclonal Antibody 7B11 on C5a-Induced EosinophilDegranulation

Eosinophil degranulation stimulated by eotaxin, RANTES, MCP-3 or C5a wasmeasured by the release of eosinophil peroxidase into the media (EPO)after stimulation with either eotaxin, RANTES, MCP-3 or C5a. EPO is aneosinophil enzyme present in eosinophilic specific granules.

The present study shows that the anti-CCR3 monoclonal antibody 7B11inhibits the eosinophil degranulation stimulated by the CCR3 chemokineseotaxin, RANTES and MCP-3, while it has no effect on eosinophildegranulation stimulated by C5a. C5a binds to a different receptor andthus it serves as a negative control.

Materials and Methods

Hank's Balanced Salt Solution (HBSS, Cat. No. 14025-092) and Dulbecco'sPhosphate Buffered Saline (PBS, Cat. No. 14190-144) were from Gibco BRL.Cytochalasin B (C-6762), Hydrogen Peroxide (H₂O₂, 3% solution, H-6520),DMSO (D-5879), Tris(hydroxymethyl)amino methane (T-1503) ando-phenylenediamine (P2903) were from Sigma Chemical Co., (St. Louis,Mo.). Polystyrene V or round bottom plates were from Costar. Purifiedhuman eosinophils were prepared as described above.

Eosinophil Degranulation Assay and Blockade by the Monoclonal Antibody7B11.

7B11 antibody solutions were prepared in PBS at 1 and 0.1 mg/ml (100×assay final concentrations). Chemokine or C5a were dissolved in HBSS, 25mM Hepes, 0.25% BSA buffer (assay buffer) at 2× assay finalconcentrations.

Eosinophils were resuspended at 2.5×10⁶/ml in assay buffer (HBSS, 25 mMHepes, 0.25% BSA). A 1:1000 volume of a 5 mg/ml cytochalasin B solutionin 100% DMSO was added to the eosinophil suspension (5 μg/ml finalconcentration). 100 μl of the cell suspension were then dispensed into96 well V bottom plates (0.25×10⁶ cells per well). 2 μl of either PBS orantibody solution (1 or 0.1 mg/ml for the 10 or 1 ug/ml final antibodyconcentrations, respectively) were added to the cells and the plateswere placed in a 37° C. incubator for 10 min. After incubation, 100 μlof chemokine solution or buffer alone were added to the cells andincubated for 30 min. After incubation the plates were centrifuged for 5min at 160 g at 10° C. After centrifugation supernatants were collectedand assayed for the presence of eosinophil peroxidase as describedbelow. Assays were normally performed in duplicate.

Eosinophil Peroxidase Assay.

Analysis of EPO concentrations were carried out following the protocoldescribed in White, S. R., et al., J. Immunol. Meth., 44:257-263 (1991)with some modifications. This assay is based on the oxidation ofo-phenylenediamine by EPO in the presence of H₂O₂. Assay finalconcentrations of substrate and H₂O₂ were 16 mM and 0.01%, respectively.The substrate stock solution (27 mM substrate, 0.016% H₂O₂) was preparedimmediately prior to use in 0.1 M Tris pH 8.0, 0.1% Triton X-100.Briefly, 75 μl of substrate solution were combined with 50 μl of samplein a flat bottom 96 well plate immediately prior to obtaining readingsat 492 nm every 15 sec for 5 min. Spectrophotometric readings wereperformed in a microplate absorbance spectrophotometer (Dynatech MR4000, Dynatech Laboratories, INC., Chantilly, Va.). Data was collectedand analyzed using the assay management software Biolinx TM version 2.1.The velocity of the reaction was calculated by interpolation betweensuccessive 3 or 4 points. Horseradish peroxidase (HRP) was used asstandard. Kinetic data were extrapolated to a standard curve obtainedwith 2, 5, 10 and 20 ng of HRP and the activity expressed in units ofEPO per million cells, with one unit corresponding to the activity thatis equivalent to the activity of 1 ng of HRP. Thus, a unit of EPO isdefined as the amount of protein that would give the same activity as 1ng of HRP.

Results

In two separate experiments, the mAb 7B11 blocked eotaxin-inducedperoxidase release from eosinophils. FIG. 23A shows that 7B11 at aconcentration of 10 μg/ml significantly inhibited the degranulationinduced by 10 or 100 nM eotaxin and by 100 nM RANTES or MCP-3.Degranulation induced by 100 nM eotaxin, RANTES or MCP-3 was inhibitedby 99, 77 and 72%, respectively. Degranulation induced by 10 nM eotaxinwas inhibited to 65% and 85% by 1 and 10 μg/ml of 7B11, respectively.Significantly, as shown in FIG. 23B, the mAb 7B11 did not inhibit thedegranulation induced by C5a.

Previous studies showed that eotaxin stimulated release of EPO parallelsthe release of other eosinophilic granule enzymes and proteins such aseosinophil cationic protein (ECP) (FIG. 24A), glucuronidase andarylsulfatase B (FIGS. 25-27). EPO, ECP and glucuronidase are present ineosinophil specific granules and arylsufatase B is present in smallgranules. Thus, eotaxin induces degranulation of both specific and smallgranules. FIGS. 25-27 and show that the eotaxin dose response curves forEPO release parallel the dose response curves for the release of othereosinophilic proteins. These assays were essentially performed asdescribed in the materials and methods for the eosinophil degranulationassay. The supernatants were then assayed for the presence of eosinophilgranule proteins using established procedures. For ECP a commerciallyavailable radioimmunoassay kit was used (Pharmacia Diagnostics, Cat. No.10-9165-01). Enzyme assays for glucuronidase and arylsulfatase B aredescribed in Kroegel, C., et al., J. of Immunol., 142:3518-3526 (1989).

EPO was the enzyme of choice in eosinophil degranulation studies becauseof convenience of assay and quantitation. EPO release by eotaxin is areflection of eosinophil degranulation in general. Because EPO releaseis paralleled by the release of other eosinophil proteins and enzymes,similarly, 7B11 blockade of degranulation induced by eotaxin as measuredby blockade of EPO release, should also be reflected in blockade ofrelease of other eosinophil proteins and enzymes.

Example 12 Basophils Express CCR3

Materials and Methods

Flow Cytometry. Cells expressing CCR3 in whole blood were identified byflow cytometry. 100 μl of heparinized whole blood was stained with 400ng of a 7B11 (anti-CCR3)-FITC preparation and 500 ng of biotin coupledanti-human IgE (PharMingen, San Diego, Calif.) in the presence of 100 μlof PBS with 0.1% azide at room temperature for 20-30 minutes. Cells werewashed once in PBS with azide and stained with 5 μl Streptavidin-QuantumRed (Sigma Immuno Chemicals, St. Louis, Mo.) for 15-30 minutes at roomtemperature. Red cells were lysed using 2 ml of an ammonium chloridelysing buffer and leukocytes were pelleted and resuspended in PBS foranalysis on a FACScan flow cytometer (Becton Dickinson, Mountainview,Calif.). Visual analysis of cells from the stained populations wasperformed after sorting cells, stained as above, using a FACSvantageflow cytometer (Becton Dickinson), and preparing Diff Quik (BaxterScientific Products, McGaw Park, II) stained slides of the collectedcells.

Enumeration of number of sites/cell. Cells were stained as above forflow cytometry. After analysis of the sample, tubes containing Quantum26 beads (Flow Cytometry Standards Corp., San Juan, PR) were used tocalibrate fluorescence. The MFSF/protein ratio for the 7B11/FITCpreparation was determined using Simply Cellular beads (Flow CytometryStandards Corp.). Median channel fluorescence of the stained cells wasthen used to calculate the mean number of bound antibody molecules/cell.

Results

The monoclonal antibody 7B11 previously shown to recognize CCR3,recognized only two populations of cells from whole blood preparations(FIG. 28). One of these populations could be shown to have high levelsof IgE on its surface, the other did not. Sorted cells lacking IgE, butstained with 7B11 were 97.3%±0.6 eosinophils as identified on slidesprepared from sorted cells. Cells bearing high levels of IgE on theirsurfaces and also stained by 7B11 appeared on stained preparations to bebasophils (92%±4.6), although the method of IgE staining, cell lysis andsorting of the samples led to degranulation of the majority of thecells. Eosinophil consistently expressed slightly higher numbers ofreceptors/cell than basophils from a given individual (p<0.001, pairedt-test). From analysis of 30 individuals, the average number of 7B11binding sites/eosinophil was found to be 24,700±5700; the average numberof sites/basophils was 19,000±4500.

Whole blood was stained with 7B11 FITC and anti-human IgE-biotinfollowed by streptavidin Quantum red and analyzed by flow cytometry.Analysis of the two color staining from whole blood indicated twopopulations bearing CCR3 (FIG. 28), one of these populations was doublestained with anti-human IgE. Comparisons of the intensity of staining byanti-CCR3 antibody indicated that eosinophils consistently stained moreintensely than basophils for expression of CCR3. Identity of cells inthese two populations was confirmed by conventional histologicalstaining and the IgE⁽⁺⁾ CCR3(+) population was found to be basophils(degranulated), while the IgE(−) CCR3(+) population was eosinophils.Forward and side scatter using backgating of the two populationsindicated that these cells light scatter properties consistent withother indications of their cell type.

Example 13 Basophil Chemotaxis to Eotaxin and MCP-4 is Blocked byAnti-CCR3 mAb

Leukocytes were obtained from unselected healthy volunteers afterinformed consent, and were isolated and fractionated by discontinuousdensity centrifugation as described (Kurimoto, Y., et al., J. Exp. Med.,170:467 (1989); Bischoff, S. C., et al., Blood, 79:2662 (1990)).Briefly, venous blood was anticoagulated with 10 mM EDTA, mixed with0.25 volume of dextran (6% in NaCl 0.9%), and erythrocytes were allowedto sediment at room temperature. After 90 min, the leukocytes werecollected and washed 3 times in HA buffer (20 mM Hepes, 125 mM NaCl, 5mM KCl, 0.5 mM glucose, 0.025% bovine serum albumin). To enrich forbasophil granulocytes, leukocytes were fractionated by Ficoll Hypaquedensity centrifugation exactly as described (Kurimoto, Y., et al., J.Exp. Med., 170:467 (1989)). Purified basophile were obtained byleukocyte fractionation by discontinuous Percoll gradient centrifugation(Bischoff, S. C., et al., Blood, 79:2662 (1990)). The basophil-rich celllayer was collected, washed in HA buffer, resuspended in 150 ul HAbuffer, and incubated for 40 min with paramagnetic beads coated with mAbagainst CD3 (12 ul), CD4 (15 ul), CD8 (12 ul), CD14 (5 ul), CD16 (5 ul)and CD19 (5 ul). The magnetically stained cell suspension was passedover a separation column placed in a strong magnetic field to eliminatecontaminating cells (MACS system, Miltenyi Biotec GmbH, BergischGladbach, FRG). The combination of Percoll gradient centrifugation andnegative selection with immunomagnetic beads yielded basophilpreparations of 80-95% purity (contaminated exclusively by smalllymphocytes) with a recovery of 30-60% (as determined by cytocentrifugeslides stained with May Gruenwald/Giemsa and measurements of totalhistamine contents). Cells were finally washed 3 times in HA buffer andresuspended in HACM buffer (HA buffer supplemented with 1 mM MgCl₂ and 1mM Cal₂).

Histamine and LTC₄ Release.

Basophil (80-180×10³/ml) in 20 mM Hepes, pH 7.4 containing 125 mMglucose and 0.025% BSA were warmed to 37° C., exposed to IL-3 (10 ng/ml)with or without anti-CCR3 (5 ug/ml) and then challenged. After 20 minthe tubes were placed on ice and histamine and LTC₄ were measured in thesupernatant (Dahinden, C. A., et al., J. Exp. Med., 179:751 (1994)).Histamine release was expressed as percent of the total content of thesample (determined after cell lysis). LTC₄ generation was expressed aspicograms LTC₄/D₄/E₄ per nanogram total histamine (which corresponds to1,000 basophils).

As shown in FIG. 29, basophils release histamine in response tochemokines, an histamine release can be blocked by MAb 7B11.

Chemotaxis

Chemokines were added to the lower wells of a 48 well chemotacticchamber (Neuro Probe, Cabin John, Md.). Cells were suspended in RPMI1640, 20 mM Hepes and 1% PPL, pH 7.4 with or without anti-CCR3 (5 ug/ml)and placed into the top wells (50,000 cells per well). Migration acrossas polycarbonate filter (polyvinyl pyrrolidone free, 5 um pore size;Nucleopore Corp., Pleasanton, Calif.) was assessed after an incubationat 37° C. in 5% CO₂ for 50 min. Migrated cells were countedmicroscopically on the lower surface of the filter after staining withMay-Gruenwald/Giemsa.

As shown in FIGS. 30A and 30B, basophils chemotax to eotaxin and MCP-4and the response is blocked with anti-CCR3 mAb 7B11.

Equivalents

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1-23. (canceled)
 24. An isolated C—C chemokine receptor 3 protein or functional portion thereof, that binds a ligand selected from the group consisting of RANTES and MCP-3.
 25. The isolated mammalian C—C chemokine receptor 3 protein or functional portion thereof of claim 24, wherein the mammalian C—C chemokine receptor 3 protein is a human C—C chemokine receptor 3 protein. 26-52. (canceled)
 53. An isolated polypeptide comprising a mammalian C—C chemokine receptor 3 protein (CCR3) or a functional portion thereof, wherein said CCR3 or functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and is encoded by a nucleic acid that hybridizes under conditions of 6×SSC containing 5× Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C., and wash conditions of 0.2×SSC, 0.5% SDS at 65° C., to a second nucleic acid selected from the group consisting of the complement of SEQ ID NO:1, the complement of the open reading frame of SEQ ID NO:1, the complement of SEQ ID NO:5, and the complement of the open reading frame of SEQ ID NO:5.
 54. The isolated polypeptide of claim 53, wherein said CCR3 or functional portion thereof mediates signaling, a cellular response, or signaling and a cellular response upon binding said ligand.
 55. The isolated polypeptide of claim 53, wherein said signaling is activation of a G protein or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca²⁺]_(i)), and said cellular response is chemotaxis, exocytosis, inflammatory mediator release or integrin activation.
 56. The isolated polypeptide of claim 53, wherein said CCR3 is encoded by SEQ ID NO:1.
 57. The isolated polypeptide of claim 53, wherein said CCR3 is encoded by SEQ ID NO:5.
 58. The isolated polypeptide of claim 53, wherein said isolated polypeptide is a fusion protein comprising said CCR3 or functional portion thereof.
 59. An isolated polypeptide comprising a mammalian C—C chemokine receptor 3 protein (CCR3) or a functional portion thereof, wherein said CCR3 protein or functional portion binds eotaxin and is encoded by a nucleic acid that hybridizes under conditions of 6×SSC containing 5× Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C., and wash conditions of 0.2×SSC, 0.5% SDS at 65° C., to a second nucleic acid selected from the group consisting of the complement of SEQ ID NO:1, the complement of the open reading frame of SEQ ID NO:1, the complement of SEQ ID NO:5, and the complement of the open reading frame of SEQ ID NO:5.
 60. The isolated polypeptide of claim 59, wherein said CCR3 or functional portion thereof mediates signaling, a cellular response, or signaling and a cellular response upon binding eotaxin.
 61. The isolated polypeptide of claim 59, wherein said signaling is activation of a G protein or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca²⁺]_(i)), and said cellular response is chemotaxis, exocytosis, inflammatory mediator release or integrin activation.
 62. The isolated polypeptide of claim 59, wherein said CCR3 is encoded by SEQ ID NO:1.
 63. The isolated polypeptide of claim 59, wherein said CCR3 is encoded by SEQ ID NO:5.
 64. The isolated polypeptide of claim 59, wherein said isolated polypeptide is a fusion protein comprising said CCR3 or functional portion thereof.
 65. An isolated polypeptide comprising a mammalian C—C chemokine receptor 3 protein (CCR3) or a functional portion thereof, wherein said CCR3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said CCR3 or functional portion thereof binds a ligand selected from the group consisting of RANTES and MCP-3.
 66. The isolated polypeptide of claim 65, wherein said CCR3 or functional portion thereof mediates signaling, a cellular response, or signaling and a cellular response upon binding said ligand.
 67. The isolated polypeptide of claim 65, wherein said signaling is activation of a G protein or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca²⁺]_(i)), and said cellular response is chemotaxis, exocytosis, inflammatory mediator release or integrin activation.
 68. The isolated polypeptide of claim 65, wherein the amino acid sequence of said CCR3 is SEQ ID NO:2.
 69. The isolated polypeptide of claim 65, wherein the amino acid sequence of said CCR3 is SEQ ID NO:6.
 70. The isolated polypeptide of claim 65, wherein said isolated polypeptide is a fusion protein comprising said CCR3 or functional portion thereof.
 71. An isolated polypeptide comprising a mammalian C—C chemokine receptor 3 protein (CCR3) or a functional portion thereof, wherein said CCR3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said CCR3 or functional portion thereof binds eotaxin.
 72. The isolated polypeptide of claim 71, wherein said CCR3 or functional portion thereof mediates signaling, a cellular response, or signaling and a cellular response upon binding eotaxin.
 73. The isolated polypeptide of claim 71, wherein said signaling is activation of a G protein or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca²⁺]_(i)), and said cellular response is chemotaxis, exocytosis, inflammatory mediator release or integrin activation.
 74. The isolated polypeptide of claim 71, wherein the amino acid sequence of said CCR3 is SEQ ID NO:2.
 75. The isolated polypeptide of claim 71, wherein the amino acid sequence of said CCR3 is SEQ ID NO:6.
 76. The isolated polypeptide of claim 71, wherein said isolated polypeptide is a fusion protein comprising said CCR3 or functional portion thereof.
 77. An isolated polypeptide comprising a C—C chemokine receptor 3 protein (CCR3), wherein the amino acid sequence of said CCR3 protein is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:6.
 78. The isolated polypeptide comprising a C—C chemokine receptor 3 protein (CCR3) of claim 77, wherein the amino acid sequence of said CCR3 protein consists of SEQ ID NO:2. 